Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

ABSTRACT

Nucleic acids and the encoded CER2-like polypeptides, At1g68440-like polypeptides or DEAD-box RNA helicase polypeptides are provided. A method of enhancing yield-related traits in plants by modulating expression of nucleic acids encoding CER2-like polypeptides or At1g68440-like polypeptides is provided. A method of enhancing yield-related traits in plants by reducing or substantially eliminating expression of nucleic acids encoding DEAD-box RNA helicase polypeptides and/or the activity of DEAD-box RNA helicase polypeptides in said plants is provided. Plants with modulated expression of the nucleic acids encoding CER2-like polypeptides or At1g68440-like polypeptides have enhanced yield-related traits relative to control plants. Plants with reduction or elimination of the expression of endogenous nucleic acids encoding DEAD-box RNA helicase polypeptides have enhanced yield-related traits relative to control plants.

The present invention relates generally to the field of molecularbiology and concerns a method for enhancing yield-related traits inplants by modulating expression in a plant of a nucleic acid encoding aDEAD-box RNA helicase polypeptide, or a CER2-like (CER2-like acyltransferase) polypeptide, or an At1g68440-like (putative IMPdehydrogenase/GMP reductase from Populus trichocarpa) polypeptide. Thepresent invention also concerns plants having modulated expression of anucleic acid encoding a DEAD-box RNA helicase polypeptide, which plantshave enhanced yield-related traits relative to corresponding wild typeplants or other control plants. The invention also provides constructsuseful in the methods of the invention.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuels research towards increasing theefficiency of agriculture. Conventional means for crop and horticulturalimprovements utilise selective breeding techniques to identify plantshaving desirable characteristics. However, such selective breedingtechniques have several drawbacks, namely that these techniques aretypically labour intensive and result in plants that often containheterogeneous genetic components that may not always result in thedesirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant. Such technology has the capacity to deliver crops or plantshaving various improved economic, agronomic or horticultural traits.

A trait of particular economic interest is increased yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Yield isdirectly dependent on several factors, for example, the number and sizeof the organs, plant architecture (for example, the number of branches),seed production, leaf senescence and more. Root development, nutrientuptake, stress tolerance and early vigour may also be important factorsin determining yield. Optimizing the abovementioned factors maytherefore contribute to increasing crop yield.

Seed yield is a particularly important trait, since the seeds of manyplants are important for human and animal nutrition. Crops such as corn,rice, wheat, canola and soybean account for over half the total humancaloric intake, whether through direct consumption of the seedsthemselves or through consumption of meat products raised on processedseeds. They are also a source of sugars, oils and many kinds ofmetabolites used in industrial processes. Seeds contain an embryo (thesource of new shoots and roots) and an endosperm (the source ofnutrients for embryo growth during germination and during early growthof seedlings). The development of a seed involves many genes, andrequires the transfer of metabolites from the roots, leaves and stemsinto the growing seed. The endosperm, in particular, assimilates themetabolic precursors of carbohydrates, oils and proteins and synthesizesthem into storage macromolecules to fill out the grain.

Another important trait for many crops is early vigour. Improving earlyvigour is an important objective of modern rice breeding programs inboth temperate and tropical rice cultivars. Long roots are important forproper soil anchorage in water-seeded rice. Where rice is sown directlyinto flooded fields, and where plants must emerge rapidly through water,longer shoots are associated with vigour. Where drill-seeding ispracticed, longer mesocotyls and coleoptiles are important for goodseedling emergence. The ability to engineer early vigour into plantswould be of great importance in agriculture. For example, poor earlyvigour has been a limitation to the introduction of maize (Zea mays L.)hybrids based on Corn Belt germplasm in the European Atlantic.

A further important trait is that of improved abiotic stress tolerance.Abiotic stress is a primary cause of crop loss worldwide, reducingaverage yields for most major crop plants by more than 50% (Wang et al.,Planta 218, 1-14, 2003). Abiotic stresses may be caused by drought,salinity, extremes of temperature, chemical toxicity and oxidativestress. The ability to improve plant tolerance to abiotic stress wouldbe of great economic advantage to farmers worldwide and would allow forthe cultivation of crops during adverse conditions and in territorieswhere cultivation of crops may not otherwise be possible.

Crop yield may therefore be increased by optimising one of theabove-mentioned factors.

Depending on the end use, the modification of certain yield traits maybe favoured over others. For example for applications such as forage orwood production, or bio-fuel resource, an increase in the vegetativeparts of a plant may be desirable, and for applications such as flour,starch or oil production, an increase in seed parameters may beparticularly desirable. Even amongst the seed parameters, some may befavoured over others, depending on the application. Various mechanismsmay contribute to increasing seed yield, whether that is in the form ofincreased seed size or increased seed number.

One approach to increasing yield (seed yield and/or biomass) in plantsmay be through modification of the inherent growth mechanisms of aplant, such as the cell cycle or various signalling pathways involved inplant growth or in defense mechanisms.

It has now been found that various yield-related traits may be improvedin plants by modulating expression in a plant of a nucleic acid encodinga DEAD-box RNA helicase polypeptide, or a CER2-like (CER2-like acyltransferase) polypeptide, or an At1g68440-like polypeptide in a plant.

BACKGROUND 1. DEAD-Box RNA Helicase Polypeptide

DEAD-box proteins play important roles in RNA metabolism and their roleseem to be very specific. In yeast, studies have shown that DEAD-boxproteins are involved in controlling temporal remodelling of theribonucleoprotein (RNP) complex. In literature, some DEAD-box proteinshave been shown to have RNA-dependent ATP-ase and ATP-dependent RNAhelicase activities in vitro. Such helicase activities require energyand allow unwinding of double stranded nucleic acids (dsDNA or dsRNA).DEAD-box and related DEAH, DExH, and DExD families, which are commonlyreferred to as the DExD/H helicase family, are members of the SF2family, and share nine highly conserved motifs (Gorbalenya and Koonin,1993; Tanner and Linder, 2001; Caruthers and McKay, 2002). These closelyrelated families can be distinguished by variations within theirconserved motifs. The DEAD-box family is by far the largest one, and ischaracterized by the presence of nine conserved motifs that are involvedin ATPase and helicase activities and in their regulation (Tanner etal., 2003). The conserved motifs of the DExD/H helicases are clusteredin a central core region that spans 350-400 amino acids (Tanner andLinder, 2001; Caruthers and McKay, 2002). By contrast, the N- andC-terminal extensions are highly variable in size and composition(Caruthers and McKay, 2002).

The DEAD-box RNA helicases form a large family of proteins found in alleukaryotes, and most prokaryotes (Aubourg et al., 1999; de la Cruz etal., 1999; Rocak and Linder, 2004). For example, nearly 30 genes thatencode DEAD-box RNA helicases were identified in genomes ofCaenorhabditis elegans and Drosophila melanogaster (Boudet et al.,2001). In Arabidopsis, >50 members of DEAD-box RNA helicases have beenidentified (Aubourg et al., 1999; Boudet et al., 2001). Aubourg et al.(1999) characterized six subfamilies of DEAD-box RNA helicases inArabidopsis. Similarities between the members of these subfamilies andknown DEAD-box RNA helicases from other organisms were found to besuggesting possible functions (Auburg et al., 1999).

Despite the involvement of DEAD-box RNA helicases in many diversebiological processes, their precise functions and regulation largelyremain to be elucidated.

2. CER2-Like Polypeptides

The vital importance of plant surface wax in protecting tissue fromenvironmental stresses is reflected in the huge commitment of epidermalcells to cuticle formation (Samuels et al. (2008). Ann. Rev. Plant Biol.59:683-707). Plants are subject to a wide range of abiotic stresses, andtheir cuticular wax layer provides a protective barrier, which consistspredominantly of long-chain hydrocarbon compounds, including alkanes,primary alcohols, aldehydes, secondary alcohols, ketones, esters andother derived compounds (Sheperd et al. (2006). New Phytol.171(3):469-99). Modification of leaf cuticular wax accumulation could beachieved by modulating expression of genes encoding different enzymes inthe pathway. In Arabidopsis, a series of so called CER mutants affectedin cuticular wax biosynthesis were identified and some of thecorresponding genes cloned. Xia and co-workers have found out that theArabidopsis CER2 gene is expressed in an organ- and tissue-specificmanner. Furthermore, CER2 is expressed at high levels only in theepidermis of young siliques and stems (Xia et al. (1996). Plant Cell.8(8):1291-304). In agreement with the activity of the CER2 promoter inhypocotyls, cuticular wax accumulates on this organ in a CER2-dependentfashion. In leaves CER2 expression is confined to the guard cells,trichomes, and petioles (Xia et al. (1997). Plant Physiol.115(3):925-37). Expression profile characterization showed developmentalregulation of wax biosynthesis. Whereas some genes like CER6 wereinduced by light and osmotic stress, surprisingly, CER2 expression inArabidopsis was unaffected by abiotic stress (Cha et al. (2007). AnalChem. 81(8):2991-3000; Cha et al. (2008). Plant J. 55(2):348-360). Brounet al. (2004, PNAS. 101(13):4706-11) demonstrated in gene expressionanalyses that a number of genes, such as CER1, KCS1, and CER2, which areknown to be involved in wax biosynthesis, were induced in WIN1, anERF-type transcription factor. Furthermore, Islam et al. showed that amodification of leaf wax accumulation alters drought response in rice.The Oryza gene GLOSSYI (GLI) is closely related to Arabidopsis CER1encoding an aldehyde decarbonylase involved in stem wax accumulation andpollen fertility (Islam et al. (2009). Plant Mol. Boil. 70(4):443-456).

3. At1g68440-Like Polypeptides

The Arabidopsis thaliana orthologue At1g68440 was identified—amongothers—as translationally regulated gene by DNA microarray analysis uponsucrose starvation of Arabidopsis cell culture (Nicolai, M., et al.Plant Physiol. 2006 June; 141(2):663-73). Comparison of transcriptionaland translational gene lists highlighted the importance of translationalregulation (mostly repression) affecting genes involved in cell cycleand cell growth, these being overrepresented in translationallyregulated genes, providing a molecular framework for the arrest of cellproliferation following starvation. Furthermore, Baxter et al. (PlantPhysiol. 2007 January; 143(1):312-25) have shown that there is adramatic change in the abundance of transcripts involved in metabolism,such as At1g68440, that serves both to mobilize alternative carbonreserves and to reconfigure metabolic fluxes to bypass some of theinhibited pathways, to cope with the resultant metabolic restrictionsupon exposure to oxidative stress. In addition, Atg68440 wasdemonstrated to be down-regulated in Arabidopsis knock-outs ofApolipoprotein D ortholog AtTIL, a member of the plant lipocalin family(Charron Jean-Benoit F, et al., BMC Plant Biol. 2008 Jul. 31; 8:86).AtTIL lipocalin is involved in modulating tolerance to oxidative stress.AtTIL knock-out plants are very sensitive to sudden drops in temperatureand paraquat treatment, and dark-grown plants die shortly after transferto light.

SUMMARY 1. DEAD-Box RNA Helicase Polypeptide

Surprisingly, it has now been found that reducing or substantiallyeliminating expression in a plant of a nucleic acid encoding anendogenous DEAD-box RNA helicase polypeptide gives plants havingenhanced yield-related traits, in particular increased seed yieldrelative to control plants.

According one embodiment, there is provided a method for improvingyield-related traits in plants relative to control plants, comprisingreducing or substantially eliminating expression in a plant of a nucleicacid encoding an endogenous DEAD-box RNA helicase polypeptide.

The section captions and headings in this specification are forconvenience and reference purpose only and should not affect in any waythe meaning or interpretation of this specification.

2. CER2-Like Polypeptides

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a CER2-like polypeptide as defined herein givesplants having enhanced yield-related traits, in particular increasedseed yield relative to control plants.

According another embodiment, there is provided a method for improvingyield-related traits as provided herein in plants relative to controlplants, comprising modulating expression in a plant of a nucleic acidencoding a CER2-like polypeptide as defined herein.

3. At1g68440-Like Polypeptides

Surprisingly, it has now been found that modulating expression of anucleic acid encoding the newly identified poplar orthologue ofAt1g68440, which therefore was named At1g68440-like polypeptide, asdefined herein gives plants having enhanced yield-related traits, inparticular increased seed yield relative to control plants.

According still another embodiment, there is provided a method forimproving yield-related traits as provided herein in plants relative tocontrol plants, comprising modulating expression in a plant of a nucleicacid encoding an At1g68440-like polypeptide as defined herein.

The section captions and headings in this specification are forconvenience and reference purpose only and should not affect in any waythe meaning or interpretation of this specification.

DEFINITIONS

The following definitions will be used throughout the presentspecification.

Polypeptide(s)/Protein(s)

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length, linkedtogether by peptide bonds.

Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/NucleotideSequence(s)

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are usedinterchangeably herein and refer to nucleotides, either ribonucleotidesor deoxyribonucleotides or a combination of both, in a polymericunbranched form of any length.

Homologue(s)

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introducedinto a predetermined site in a protein. Insertions may compriseN-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than N- or C-terminalfusions, of the order of about 1 to 10 residues. Examples of N- orC-terminal fusion proteins or peptides include the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)-6-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

A substitution refers to replacement of amino acids of the protein withother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide andmay range from 1 to 10 amino acids; insertions will usually be of theorder of about 1 to 10 amino acid residues. The amino acid substitutionsare preferably conservative amino acid substitutions. Conservativesubstitution tables are well known in the art (see for example Creighton(1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).

TABLE 1 Examples of conserved amino acid substitutions ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln AsnCys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg;Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr TyrTrp; Phe Val Ile; Leu

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation. Methods for the manipulation of DNA sequences to producesubstitution, insertion or deletion variants of a protein are well knownin the art. For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols (see Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989 and yearly updates)).

Derivatives

“Derivatives” include peptides, oligopeptides, polypeptides which may,compared to the amino acid sequence of the naturally-occurring form ofthe protein, such as the protein of interest, comprise substitutions ofamino acids with non-naturally occurring amino acid residues, oradditions of non-naturally occurring amino acid residues. “Derivatives”of a protein also encompass peptides, oligopeptides, polypeptides whichcomprise naturally occurring altered (glycosylated, acylated,prenylated, phosphorylated, myristoylated, sulphated etc.) ornon-naturally altered amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents or additionscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein. Furthermore, “derivatives” also includefusions of the naturally-occurring form of the protein with taggingpeptides such as FLAG, HIS6 or thioredoxin (for a review of taggingpeptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Orthologue(s)/Paralogue(s)

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

Domain, Motif/Consensus Sequence/Signature

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

Specialist databases exist for the identification of domains, forexample, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Batemanet al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of toolsfor in silico analysis of protein sequences is available on the ExPASyproteomics server (Swiss Institute of Bioinformatics (Gasteiger et al.,ExPASy: the proteomics server for in-depth protein knowledge andanalysis, Nucleic Acids Res. 31:3784-3788 (2003)). Domains or motifs mayalso be identified using routine techniques, such as by sequencealignment.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information(NCBI). Homologues may readily be identified using, for example, theClustalW multiple sequence alignment algorithm (version 1.83), with thedefault pairwise alignment parameters, and a scoring method inpercentage. Global percentages of similarity and identity may also bedetermined using one of the methods available in the MatGAT softwarepackage (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29.MatGAT: an application that generates similarity/identity matrices usingprotein or DNA sequences). Minor manual editing may be performed tooptimise alignment between conserved motifs, as would be apparent to aperson skilled in the art. Furthermore, instead of using full-lengthsequences for the identification of homologues, specific domains mayalso be used. The sequence identity values may be determined over theentire nucleic acid or amino acid sequence or over selected domains orconserved motif(s), using the programs mentioned above using the defaultparameters. For local alignments, the Smith-Waterman algorithm isparticularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1);195-7).

Reciprocal BLAST

Typically, this involves a first BLAST involving BLASTing a querysequence (for example using any of the sequences listed in Table A ofthe Examples section) against any sequence database, such as thepublicly available NCBI database. BLASTN or TBLASTX (using standarddefault values) are generally used when starting from a nucleotidesequence, and BLASTP or TBLASTN (using standard default values) whenstarting from a protein sequence. The BLAST results may optionally befiltered. The full-length sequences of either the filtered results ornon-filtered results are then BLASTed back (second BLAST) againstsequences from the organism from which the query sequence is derived.The results of the first and second BLASTs are then compared. Aparalogue is identified if a high-ranking hit from the first blast isfrom the same species as from which the query sequence is derived, aBLAST back then ideally results in the query sequence amongst thehighest hits; an orthologue is identified if a high-ranking hit in thefirst BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Hybridisation

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. The hybridisation process can furthermore occur with one ofthe complementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which ahybridisation takes place. The stringency of hybridisation is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridisation buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Medium stringency conditions are when the temperatureis 20° C. below T_(m), and high stringency conditions are when thetemperature is 10° C. below T_(m). High stringency hybridisationconditions are typically used for isolating hybridising sequences thathave high sequence similarity to the target nucleic acid sequence.However, nucleic acids may deviate in sequence and still encode asubstantially identical polypeptide, due to the degeneracy of thegenetic code. Therefore medium stringency hybridisation conditions maysometimes be needed to identify such nucleic acid molecules.

The T_(m) is the temperature under defined ionic strength and pH, atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. The T_(m) is dependent upon the solution conditions and the basecomposition and length of the probe. For example, longer sequenceshybridise specifically at higher temperatures. The maximum rate ofhybridisation is obtained from about 16° C. up to 32° C. below T_(m).The presence of monovalent cations in the hybridisation solution reducethe electrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M (for higher concentrations, this effect maybe ignored). Formamide reduces the melting temperature of DNA-DNA andDNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, andaddition of 50% formamide allows hybridisation to be performed at 30 to45° C., though the rate of hybridisation will be lowered. Base pairmismatches reduce the hybridisation rate and the thermal stability ofthe duplexes. On average and for large probes, the Tm decreases about 1°C. per % base mismatch. The T_(m) may be calculated using the followingequations, depending on the types of hybrids:

1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284,1984):

T _(m)=81.5° C.+16.6×log₁₀[Na⁺]^(a)+0.41×%[G/C ^(b)]−500×[L^(c)]⁻¹−0.61×% formamide

2) DNA-RNA or RNA-RNA hybrids:

T _(m)=79.8° C.+18.5(log₁₀[Na⁺]^(a))+0.58(%G/C ^(b))+11.8(%G/C_(b))²−820/L ^(c)

3) oligo-DNA or oligo-RNAs hybrids:

For <20 nucleotides: T _(m)=2(l _(n))

For 20-35 nucleotides: T _(m)=22+1.46(l _(n))

^(a) or for other monovalent cation, but only accurate in the 0.01-0.4 Mrange.^(b) only accurate for % GC in the 30% to 75% range.^(c) L=length of duplex in base pairs.^(d) oligo, oligonucleotide; l_(n), =effective length of primer=2×(no.of G/C)+(no. of A/T).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase. Fornon-homologous probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridisation and which will either maintain or change the stringencyconditions.

Besides the hybridisation conditions, specificity of hybridisationtypically also depends on the function of post-hybridisation washes. Toremove background resulting from non-specific hybridisation, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridisation stringency. A positive hybridisationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridisationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridisation conditions for DNAhybrids longer than 50 nucleotides encompass hybridisation at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridisationconditions for DNA hybrids longer than 50 nucleotides encompasshybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridising nucleic acid. Whennucleic acids of known sequence are hybridised, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridisation solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

Splice Variant

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Such variants will be ones in which thebiological activity of the protein is substantially retained; this maybe achieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor predicting and isolating such splice variants are well known in theart (see for example Foissac and Schiex (2005) BMC Bioinformatics 6:25).

Allelic Variant

Alleles or allelic variants are alternative forms of a given gene,located at the same chromosomal position. Allelic variants encompassSingle Nucleotide Polymorphisms (SNPs), as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.

Endogenous Gene

Reference herein to an “endogenous” gene not only refers to the gene inquestion as found in a plant in its natural form (i.e., without therebeing any human intervention), but also refers to that same gene (or asubstantially homologous nucleic acid/gene) in an isolated formsubsequently (re)introduced into a plant (a transgene). For example, atransgenic plant containing such a transgene may encounter a substantialreduction of the transgene expression and/or substantial reduction ofexpression of the endogenous gene. The isolated gene may be isolatedfrom an organism or may be manmade, for example by chemical synthesis.

Gene Shuffling/Directed Evolution

Gene shuffling or directed evolution consists of iterations of DNAshuffling followed by appropriate screening and/or selection to generatevariants of nucleic acids or portions thereof encoding proteins having amodified biological activity (Castle et al., (2004) Science 304(5674):1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

Construct

Artificial DNA (such as but, not limited to plasmids or viral DNA)capable of replication in a host cell and used for introduction of a DNAsequence of interest into a host cell or host organism. Host cells ofthe invention may be any cell selected from bacterial cells, such asEscherichia coli or Agrobacterium species cells, yeast cells, fungal,algal or cyanobacterial cells or plant cells. The skilled artisan iswell aware of the genetic elements that must be present on the geneticconstruct in order to successfully transform, select and propagate hostcells containing the sequence of interest. The sequence of interest isoperably linked to one or more control sequences (at least to apromoter) as described herein. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Thoseskilled in the art will be aware of terminator and enhancer sequencesthat may be suitable for use in performing the invention. An intronsequence may also be added to the 5′ untranslated region (UTR) or in thecoding sequence to increase the amount of the mature message thataccumulates in the cytosol, as described in the definitions section.Other control sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements. Such sequences would be known or may readily beobtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene. Selectable markers aredescribed in more detail in the “definitions” section herein. The markergenes may be removed or excised from the transgenic cell once they areno longer needed. Techniques for marker removal are known in the art,useful techniques are described above in the definitions section.

Regulatory Element/Control Sequence/Promoter

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are ligated. The term“promoter” typically refers to a nucleic acid control sequence locatedupstream from the transcriptional start of a gene and which is involvedin recognising and binding of RNA polymerase and other proteins, therebydirecting transcription of an operably linked nucleic acid. Encompassedby the aforementioned terms are transcriptional regulatory sequencesderived from a classical eukaryotic genomic gene (including the TATA boxwhich is required for accurate transcription initiation, with or withouta CCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative that confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ.

A “plant promoter” comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or comprise asuitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern.

For the identification of functionally equivalent promoters, thepromoter strength and/or expression pattern of a candidate promoter maybe analysed for example by operably linking the promoter to a reportergene and assaying the expression level and pattern of the reporter genein various tissues of the plant. Suitable well-known reporter genesinclude for example beta-glucuronidase or beta-galactosidase. Thepromoter activity is assayed by measuring the enzymatic activity of thebeta-glucuronidase or beta-galactosidase. The promoter strength and/orexpression pattern may then be compared to that of a reference promoter(such as the one used in the methods of the present invention).Alternatively, promoter strength may be assayed by quantifying mRNAlevels or by comparing mRNA levels of the nucleic acid used in themethods of the present invention, with mRNA levels of housekeeping genessuch as 18S rRNA, using methods known in the art, such as Northernblotting with densitometric analysis of autoradiograms, quantitativereal-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).Generally by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts, to about 1/500,0000 transcripts per cell. Conversely, a“strong promoter” drives expression of a coding sequence at high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000transcripts per cell. Generally, by “medium strength promoter” isintended a promoter that drives expression of a coding sequence at alower level than a strong promoter, in particular at a level that is inall instances below that obtained when under the control of a 35S CaMVpromoter.

Operably Linked

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

Constitutive Promoter

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Table 2a below gives examples of constitutivepromoters.

TABLE 2a Examples of constitutive promoters Gene Source Reference ActinMcElroy et al, Plant Cell, 2: 163-171, 1990 HMGP WO 2004/070039 CAMV 35SOdell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant J Nov;2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen et al, PlantMol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al, Plant MolBiol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol. Gen.Genet. 231: 276-285, 1992 Alfalfa H3 Wu et al. Plant Mol. Biol. 11:641-649, 1988 histone Actin 2 An et al, Plant J. 10(1); 107-121, 199634S FMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubiscosmall U.S. Pat. No. 4,962,028 subunit OCS Leisner (1988) Proc Natl AcadSci USA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696SAD2 Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al.(1984) Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Superpromoter WO 95/14098 G-box proteins WO 94/12015

Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells ofan organism.

Developmentally-Regulated Promoter

A developmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges.

Inducible Promoter

An inducible promoter has induced or increased transcription initiationin response to a chemical (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108), environmental or physicalstimulus, or may be “stress-inducible”, i.e. activated when a plant isexposed to various stress conditions, or a “pathogen-inducible” i.e.activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”.

Examples of root-specific promoters are listed in Table 2b below:

TABLE 2b Examples of root-specific promoters Gene Source Reference RCc3Plant Mol Biol. 1995 January; 27(2): 237-48 Arabidopsis PHT1 Koyama etal. J Biosci Bioeng. 2005 January; 99(1): 38-42.; Mudge et al. (2002,Plant J. 31: 341) Medicago phosphate Xiao et al., 2006, Plant Biol(Stuttg). 2006 transporter July; 8(4): 439-49 Arabidopsis Pyk10 Nitz etal. (2001) Plant Sci 161(2): 337-346 root-expressible genes Tingey etal., EMBO J. 6: 1, 1987. tobacco auxin- Van der Zaal et al., Plant Mol.Biol. 16, 983, inducible gene 1991. β-tubulin Oppenheimer, et al., Gene63: 87, 1988. tobacco root- Conkling, et al., Plant Physiol. 93: 1203,1990. specific genes B. napus G1-3b gene U.S. Pat. No. 5,401,836 SbPRP1Suzuki et al., Plant Mol. Biol. 21: 109-119, 1993. LRX1 Baumberger etal. 2001, Genes & Dev. 15: 1128 BTG-26 Brassica US 20050044585 napusLeAMT1 (tomato) Lauter et al. (1996, PNAS 3: 8139) The LeNRT1-1 Lauteret al. (1996, PNAS 3: 8139) (tomato) class I patatin Liu et al., PlantMol. Biol. 17 (6): 1139-1154 gene (potato) KDC1 (Daucus Downey et al.(2000, J. Biol. Chem. 275: 39420) carota) TobRB7 gene W Song (1997) PhDThesis, North Carolina State University, Raleigh, NC USA OsRAB5a (rice)Wang et al. 2002, Plant Sci. 163: 273 ALF5 (Arabidopsis) Diener et al.(2001, Plant Cell 13: 1625) NRT2; 1Np (N. Quesada et al. (1997, PlantMol. Biol. 34: 265) plumbaginifolia)

A seed-specific promoter is transcriptionally active predominantly inseed tissue, but not necessarily exclusively in seed tissue (in cases ofleaky expression). The seed-specific promoter may be active during seeddevelopment and/or during germination. The seed specific promoter may beendosperm/aleurone/embryo specific. Examples of seed-specific promoters(endosperm/aleurone/embryo specific) are shown in Table 2c to Table 2fbelow. Further examples of seed-specific promoters are given in Qing Quand Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure isincorporated by reference herein as if fully set forth.

TABLE 2c Examples of seed-specific promoters Gene source Referenceseed-specific genes Simon et al., Plant Mol. Biol. 5: 191, 1985;Scofield et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin Pearson et al., PlantMol. Biol. 18: 235-245, 1992. legumin Ellis et al., Plant Mol. Biol. 10:203-214, 1988. glutelin (rice) Takaiwa et al., Mol. Gen. Genet. 208:15-22, 1986; Takaiwa et al., FEBS Letts. 221: 43-47, 1987. zein Matzkeet al Plant Mol Biol, 14(3): 323-32 1990 napA Stalberg et al, Planta199: 515-519, 1996. wheat LMW and HMW Mol Gen Genet 216: 81-90, 1989;NAR 17: 461-2, 1989 glutenin-1 wheat SPA Albani et al, Plant Cell, 9:171-184, 1997 wheat α, β, γ-gliadins EMBO J. 3: 1409-15, 1984 barleyItr1 promoter Diaz et al. (1995) Mol Gen Genet 248(5): 592-8 barley B1,C, D, hordein Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993;Mol Gen Genet 250: 750-60, 1996 barley DOF Mena et al, The PlantJournal, 116(1): 53-62, 1998 blz2 EP99106056.7 synthetic promoterVicente-Carbajosa et al., Plant J. 13: 629-640, 1998. rice prolaminNRP33 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 ricea-globulin Glb-1 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998rice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996rice α-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33: 513-522,1997 rice ADP-glucose pyrophos- Trans Res 6: 157-68, 1997 phorylasemaize ESR gene family Plant J 12: 235-46, 1997 sorghum α-kafirin DeRoseet al., Plant Mol. Biol 32: 1029-35, 1996 KNOX Postma-Haarsma et al,Plant Mol. Biol. 39: 257-71, 1999 rice oleosin Wu et al, J. Biochem.123: 386, 1998 sunflower oleosin Cummins et al., Plant Mol. Biol. 19:873-876, 1992 PRO0117, putative rice 40S WO 2004/070039 ribosomalprotein PRO0136, rice alanine unpublished aminotransferase PRO0147,trypsin inhibitor unpublished ITR1 (barley) PRO0151, rice WSI18 WO2004/070039 PRO0175, rice RAB21 WO 2004/070039 PRO005 WO 2004/070039PRO0095 WO 2004/070039 α-amylase (Amy32b) Lanahan et al, Plant Cell 4:203-211, 1992; Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270, 1991cathepsin β-like gene Cejudo et al, Plant Mol Biol 20: 849-856, 1992Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 Chi26 Leah et al.,Plant J. 4: 579-89, 1994 Maize B-Peru Selinger et al., Genetics 149;1125-38, 1998

TABLE 2d examples of endosperm-specific promoters Gene source Referenceglutelin (rice) Takaiwa et al. (1986) Mol Gen Genet 208: 15-22; Takaiwaet al. (1987) FEBS Letts. 221: 43-47 zein Matzke et al., (1990) PlantMol Biol 14(3): 323-32 wheat LMW and Colot et al. (1989) Mol Gen Genet216: 81-90, HMW glutenin-1 Anderson et al. (1989) NAR 17: 461-2 wheatSPA Albani et al. (1997) Plant Cell 9: 171-184 wheat gliadins Rafalskiet al. (1984) EMBO 3: 1409-15 barley Itr1 promoter Diaz et al. (1995)Mol Gen Genet 248(5): 592-8 barley B1, C, D, Cho et al. (1999) TheorAppl Genet 98: 1253-62; hordein Muller et al. (1993) Plant J 4: 343-55;Sorenson et al. (1996) Mol Gen Genet 250: 750-60 barley DOF Mena et al,(1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem274(14): 9175-82 synthetic promoter Vicente-Carbajosa et al. (1998)Plant J 13: 629-640 rice prolamin Wu et al, (1998) Plant Cell Physiol39(8) 885-889 NRP33 rice globulin Glb-1 Wu et al. (1998) Plant CellPhysiol 39(8) 885-889 rice globulin Nakase et al. (1997) Plant MolecBiol 33: 513-522 REB/OHP-1 rice ADP-glucose Russell et al. (1997) TransRes 6: 157-68 pyrophosphorylase maize ESR gene Opsahl-Ferstad et al.(1997) Plant J 12: 235-46 family sorghum kafirin DeRose et al. (1996)Plant Mol Biol 32: 1029-35

TABLE 2e Examples of embryo specific promoters: Gene source Referencerice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 PRO0151 WO2004/070039 PRO0175 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO2004/070039

TABLE 2f Examples of aleurone-specific promoters: Gene source Referenceα-amylase Lanahan et al, Plant Cell 4: 203-211, 1992; (Amy32b) Skriveret al, Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin β-like geneCejudo et al, Plant Mol Biol 20: 849-856, 1992 Barley Ltp2 Kalla et al.,Plant J. 6: 849-60, 1994 Chi26 Leah et al., Plant J. 4: 579-89, 1994Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998

A green tissue-specific promoter as defined herein is a promoter that istranscriptionally active predominantly in green tissue, substantially tothe exclusion of any other parts of a plant, whilst still allowing forany leaky expression in these other plant parts.

Examples of green tissue-specific promoters which may be used to performthe methods of the invention are shown in Table 2g below.

TABLE 2g Examples of green tissue-specific promoters Gene ExpressionReference Maize Orthophosphate Leaf specific Fukavama et al., Plantdikinase Physiol. 2001 November; 127(3): 1136-46 Maize Phosphoenol- Leafspecific Kausch et al., Plant Mol Biol. pyruvate carboxylase 2001January; 45(1): 1-15 Rice Phosphoenol- Leaf specific Lin et al., 2004DNA Seq. 2004 pyruvate carboxylase August; 15(4): 269-76 Rice smallsubunit Leaf specific Nomura et al., Plant Mol Biol. Rubisco 2000September; 44(1): 99-106 rice beta expansin Shoot specific WO2004/070039 EXBP9 Pigeonpea small Leaf specific Panguluri et al., IndianJ Exp subunit Biol. 2005 April; 43(4): 369-72 Rubisco Pea RBCS3A Leafspecific

Another example of a tissue-specific promoter is a meristem-specificpromoter, which is transcriptionally active predominantly inmeristematic tissue, substantially to the exclusion of any other partsof a plant, whilst still allowing for any leaky expression in theseother plant parts. Examples of green meristem-specific promoters whichmay be used to perform the methods of the invention are shown in Table2h below.

TABLE 2h Examples of meristem-specific promoters Gene source Expressionpattern Reference rice OSH1 Shoot apical meristem, Sato et al. (1996)from embryo globular Proc. Natl. Acad. Sci. stage to seedling stage USA,93: 8117-8122 Rice metallothionein Meristem specific BAD87835.1 WAK1 &WAK 2 Shoot and root apical Wagner & Kohorn meristems, and in ex- (2001)Plant Cell panding leaves and sepals 13(2): 303-318

Terminator

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

Selectable Marker (Gene)/Reporter Gene

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS or β-galactosidasewith its coloured substrates, for example X-Gal), luminescence (such asthe luciferin/luceferase system) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof). This list represents only asmall number of possible markers. The skilled worker is familiar withsuch markers. Different markers are preferred, depending on the organismand the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die).

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible. Naturally, these methods can also be applied tomicroorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

-   -   (a) the nucleic acid sequences encoding proteins useful in the        methods of the invention, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence according to the invention, for        example a promoter, or    -   (c) a) and b)        are not located in their natural genetic environment or have        been modified by recombinant methods, it being possible for the        modification to take the form of, for example, a substitution,        addition, deletion, inversion or insertion of one or more        nucleotide residues. The natural genetic environment is        understood as meaning the natural genomic or chromosomal locus        in the original plant or the presence in a genomic library. In        the case of a genomic library, the natural genetic environment        of the nucleic acid sequence is preferably retained, at least in        part. The environment flanks the nucleic acid sequence at least        on one side and has a sequence length of at least 50 bp,        preferably at least 500 bp, especially preferably at least 1000        bp, most preferably at least 5000 bp. A naturally occurring        expression cassette—for example the naturally occurring        combination of the natural promoter of the nucleic acid        sequences with the corresponding nucleic acid sequence encoding        a polypeptide useful in the methods of the present invention, as        defined above—becomes a transgenic expression cassette when this        expression cassette is modified by non-natural, synthetic        (“artificial”) methods such as, for example, mutagenic        treatment. Suitable methods are described, for example, in U.S.        Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not present in, or originating from, the genome of saidplant, or are present in the genome of said plant but not at theirnatural locus in the genome of said plant, it being possible for thenucleic acids to be expressed homologously or heterologously. However,as mentioned, transgenic also means that, while the nucleic acidsaccording to the invention or used in the inventive method are at theirnatural position in the genome of a plant, the sequence has beenmodified with regard to the natural sequence, and/or that the regulatorysequences of the natural sequences have been modified. Transgenic ispreferably understood as meaning the expression of the nucleic acidsaccording to the invention at an unnatural locus in the genome, i.e.homologous or, preferably, heterologous expression of the nucleic acidstakes place. Preferred transgenic plants are mentioned herein.

It shall further be noted that in the context of the present invention,the term “isolated nucleic acid” or “isolated polypeptide” may in someinstances be considered as a synonym for a “recombinant nucleic acid” ora “recombinant polypeptide”, respectively and refers to a nucleic acidor polypeptide that is not located in its natural genetic environmentand/or that has been modified by recombinant methods.

Modulation

The term “modulation” means in relation to expression or geneexpression, a process in which the expression level is changed by saidgene expression in comparison to the control plant, the expression levelmay be increased or decreased. The original, unmodulated expression maybe of any kind of expression of a structural RNA (rRNA, tRNA) or mRNAwith subsequent translation. For the purposes of this invention, theoriginal unmodulated expression may also be absence of any expression.The term “modulating the activity” shall mean any change of theexpression of the inventive nucleic acid sequences or encoded proteins,which leads to increased yield and/or increased growth of the plants.The expression can increase from zero (absence of, or immeasurableexpression) to a certain amount, or can decrease from a certain amountto immeasurable small amounts or zero.

Expression

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic construct. The term“expression” or “gene expression” in particular means the transcriptionof a gene or genes or genetic construct into structural RNA (rRNA, tRNA)or mRNA with or without subsequent translation of the latter into aprotein. The process includes transcription of DNA and processing of theresulting mRNA product.

Increased Expression/Overexpression

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level. For the purposes of this invention, the originalwild-type expression level might also be zero, i.e. absence ofexpression or immeasurable expression.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the polypeptide ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Decreased Expression

Reference herein to “decreased expression” or “reduction or substantialelimination” of expression is taken to mean a decrease in endogenousgene expression and/or polypeptide levels and/or polypeptide activityrelative to control plants. The reduction or substantial elimination isin increasing order of preference at least 10%, 20%, 30%, 40% or 50%,60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reducedcompared to that of control plants.

For the reduction or substantial elimination of expression an endogenousgene in a plant, a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence is required. In order to performgene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10 or fewer nucleotides, alternatively this may be as much asthe entire gene (including the 5′ and/or 3′ UTR, either in part or inwhole). The stretch of substantially contiguous nucleotides may bederived from the nucleic acid encoding the protein of interest (targetgene), or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest. Preferably, thestretch of substantially contiguous nucleotides is capable of forminghydrogen bonds with the target gene (either sense or antisense strand),more preferably, the stretch of substantially contiguous nucleotideshas, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene(either sense or antisense strand). A nucleic acid sequence encoding a(functional) polypeptide is not a requirement for the various methodsdiscussed herein for the reduction or substantial elimination ofexpression of an endogenous gene.

This reduction or substantial elimination of expression may be achievedusing routine tools and techniques. A preferred method for the reductionor substantial elimination of endogenous gene expression is byintroducing and expressing in a plant a genetic construct into which thenucleic acid (in this case a stretch of substantially contiguousnucleotides derived from the gene of interest, or from any nucleic acidcapable of encoding an orthologue, paralogue or homologue of any one ofthe protein of interest) is cloned as an inverted repeat (in part orcompletely), separated by a spacer (non-coding DNA).

In such a preferred method, expression of the endogenous gene is reducedor substantially eliminated through RNA-mediated silencing using aninverted repeat of a nucleic acid or a part thereof (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), preferably capableof forming a hairpin structure. The inverted repeat is cloned in anexpression vector comprising control sequences. A non-coding DNA nucleicacid sequence (a spacer, for example a matrix attachment region fragment(MAR), an intron, a polylinker, etc.) is located between the twoinverted nucleic acids forming the inverted repeat. After transcriptionof the inverted repeat, a chimeric RNA with a self-complementarystructure is formed (partial or complete). This double-stranded RNAstructure is referred to as the hairpin RNA (hpRNA). The hpRNA isprocessed by the plant into siRNAs that are incorporated into anRNA-induced silencing complex (RISC). The RISC further cleaves the mRNAtranscripts, thereby substantially reducing the number of mRNAtranscripts to be translated into polypeptides. For further generaldetails see for example, Grierson et al. (1998) WO 98/53083; Waterhouseet al. (1999) WO 99/53050).

Performance of the methods of the invention does not rely on introducingand expressing in a plant a genetic construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of severalwell-known “gene silencing” methods may be used to achieve the sameeffects.

One such method for the reduction of endogenous gene expression isRNA-mediated silencing of gene expression (downregulation). Silencing inthis case is triggered in a plant by a double stranded RNA sequence(dsRNA) that is substantially similar to the target endogenous gene.This dsRNA is further processed by the plant into about 20 to about 26nucleotides called short interfering RNAs (siRNAs). The siRNAs areincorporated into an RNA-induced silencing complex (RISC) that cleavesthe mRNA transcript of the endogenous target gene, thereby substantiallyreducing the number of mRNA transcripts to be translated into apolypeptide. Preferably, the double stranded RNA sequence corresponds toa target gene.

Another example of an RNA silencing method involves the introduction ofnucleic acid sequences or parts thereof (in this case a stretch ofsubstantially contiguous nucleotides derived from the gene of interest,or from any nucleic acid capable of encoding an orthologue, paralogue orhomologue of the protein of interest) in a sense orientation into aplant. “Sense orientation” refers to a DNA sequence that is homologousto an mRNA transcript thereof. Introduced into a plant would thereforebe at least one copy of the nucleic acid sequence. The additionalnucleic acid sequence will reduce expression of the endogenous gene,giving rise to a phenomenon known as co-suppression. The reduction ofgene expression will be more pronounced if several additional copies ofa nucleic acid sequence are introduced into the plant, as there is apositive correlation between high transcript levels and the triggeringof co-suppression.

Another example of an RNA silencing method involves the use of antisensenucleic acid sequences. An “antisense” nucleic acid sequence comprises anucleotide sequence that is complementary to a “sense” nucleic acidsequence encoding a protein, i.e. complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA transcriptsequence. The antisense nucleic acid sequence is preferablycomplementary to the endogenous gene to be silenced. The complementaritymay be located in the “coding region” and/or in the “non-coding region”of a gene. The term “coding region” refers to a region of the nucleotidesequence comprising codons that are translated into amino acid residues.The term “non-coding region” refers to 5′ and 3′ sequences that flankthe coding region that are transcribed but not translated into aminoacids (also referred to as 5′ and 3′ untranslated regions).

Antisense nucleic acid sequences can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid sequencemay be complementary to the entire nucleic acid sequence (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), but may also be anoligonucleotide that is antisense to only a part of the nucleic acidsequence (including the mRNA 5′ and 3′ UTR). For example, the antisenseoligonucleotide sequence may be complementary to the region surroundingthe translation start site of an mRNA transcript encoding a polypeptide.The length of a suitable antisense oligonucleotide sequence is known inthe art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10nucleotides in length or less. An antisense nucleic acid sequenceaccording to the invention may be constructed using chemical synthesisand enzymatic ligation reactions using methods known in the art. Forexample, an antisense nucleic acid sequence (e.g., an antisenseoligonucleotide sequence) may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acid sequences, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides may be used. Examples of modified nucleotidesthat may be used to generate the antisense nucleic acid sequences arewell known in the art. Known nucleotide modifications includemethylation, cyclization and ‘caps’ and substitution of one or more ofthe naturally occurring nucleotides with an analogue such as inosine.Other modifications of nucleotides are well known in the art.

The antisense nucleic acid sequence can be produced biologically usingan expression vector into which a nucleic acid sequence has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Preferably, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an operably linked antisenseoligonucleotide, and a terminator.

The nucleic acid molecules used for silencing in the methods of theinvention (whether introduced into a plant or generated in situ)hybridize with or bind to mRNA transcripts and/or genomic DNA encoding apolypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid sequence which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introducedinto a plant by transformation or direct injection at a specific tissuesite. Alternatively, antisense nucleic acid sequences can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense nucleic acid sequences can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid sequence to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid sequences canalso be delivered to cells using the vectors described herein.

According to a further aspect, the antisense nucleic acid sequence is ana-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequenceforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual b-units, the strands run parallel to each other(Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisensenucleic acid sequence may also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).

The reduction or substantial elimination of endogenous gene expressionmay also be performed using ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid sequence, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can beused to catalytically cleave mRNA transcripts encoding a polypeptide,thereby substantially reducing the number of mRNA transcripts to betranslated into a polypeptide. A ribozyme having specificity for anucleic acid sequence can be designed (see for example: Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, mRNA transcripts corresponding to a nucleic acid sequencecan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (Bartel and Szostak (1993) Science261, 1411-1418). The use of ribozymes for gene silencing in plants isknown in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by strategies asdescribed by, among others, Angell and Baulcombe ((1999) Plant J 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

Gene silencing may also occur if there is a mutation on an endogenousgene and/or a mutation on an isolated gene/nucleic acid subsequentlyintroduced into a plant. The reduction or substantial elimination may becaused by a non-functional polypeptide. For example, the polypeptide maybind to various interacting proteins; one or more mutation(s) and/ortruncation(s) may therefore provide for a polypeptide that is still ableto bind interacting proteins (such as receptor proteins) but that cannotexhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acidsequences complementary to the regulatory region of the gene (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the gene in target cells. See Helene, C.,Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad.Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.

Other methods, such as the use of antibodies directed to an endogenouspolypeptide for inhibiting its function in planta, or interference inthe signalling pathway in which a polypeptide is involved, will be wellknown to the skilled man. In particular, it can be envisaged thatmanmade molecules may be useful for inhibiting the biological functionof a target polypeptide, or for interfering with the signalling pathwayin which the target polypeptide is involved.

Alternatively, a screening program may be set up to identify in a plantpopulation natural variants of a gene, which variants encodepolypeptides with reduced activity. Such natural variants may also beused for example, to perform homologous recombination.

Artificial and/or natural microRNAs (miRNAs) may be used to knock outgene expression and/or mRNA translation. Endogenous miRNAs are singlestranded small RNAs of typically 19-24 nucleotides long. They functionprimarily to regulate gene expression and/or mRNA translation. Mostplant microRNAs (miRNAs) have perfect or near-perfect complementaritywith their target sequences. However, there are natural targets with upto five mismatches. They are processed from longer non-coding RNAs withcharacteristic fold-back structures by double-strand specific RNases ofthe Dicer family. Upon processing, they are incorporated in theRNA-induced silencing complex (RISC) by binding to its main component,an Argonaute protein. mRNAs serve as the specificity components of RISC,since they base-pair to target nucleic acids, mostly mRNAs, in thecytoplasm. Subsequent regulatory events include target mRNA cleavage anddestruction and/or translational inhibition. Effects of miRNAoverexpression are thus often reflected in decreased mRNA levels oftarget genes.

Artificial microRNAs (amiRNAs), which are typically 21 nucleotides inlength, can be genetically engineered specifically to negativelyregulate gene expression of single or multiple genes of interest.Determinants of plant microRNA target selection are well known in theart. Empirical parameters for target recognition have been defined andcan be used to aid in the design of specific amiRNAs, (Schwab et al.,Dev. Cell 8, 517-527, 2005). Convenient tools for design and generationof amiRNAs and their precursors are also available to the public (Schwabet al., Plant Cell 18, 1121-1133, 2006).

For optimal performance, the gene silencing techniques used for reducingexpression in a plant of an endogenous gene requires the use of nucleicacid sequences from monocotyledonous plants for transformation ofmonocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleic acidsequence from any given plant species is introduced into that samespecies. For example, a nucleic acid sequence from rice is transformedinto a rice plant. However, it is not an absolute requirement that thenucleic acid sequence to be introduced originates from the same plantspecies as the plant in which it will be introduced. It is sufficientthat there is substantial homology between the endogenous target geneand the nucleic acid to be introduced.

Described above are examples of various methods for the reduction orsubstantial elimination of expression in a plant of an endogenous gene.A person skilled in the art would readily be able to adapt theaforementioned methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

Transformation

The term “introduction” or “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. Plant tissue capableof subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen. Genet. 202: 179-185); DNAor RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis (Arabidopsis thaliana is within thescope of the present invention not considered as a crop plant), or cropplants such as, by way of example, tobacco plants, for example byimmersing bruised leaves or chopped leaves in an agrobacterial solutionand then culturing them in suitable media. The transformation of plantsby means of Agrobacterium tumefaciens is described, for example, byHöfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is knowninter alia from F. F. White, Vectors for Gene Transfer in Higher Plants;in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet. 208:1-9; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe inflorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent A F(1998) The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the above-mentioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

T-DNA Activation Tagging

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),involves insertion of T-DNA, usually containing a promoter (may also bea translation enhancer or an intron), in the genomic region of the geneof interest or 10 kb up- or downstream of the coding region of a gene ina configuration such that the promoter directs expression of thetargeted gene. Typically, regulation of expression of the targeted geneby its natural promoter is disrupted and the gene falls under thecontrol of the newly introduced promoter. The promoter is typicallyembedded in a T-DNA. This T-DNA is randomly inserted into the plantgenome, for example, through Agrobacterium infection and leads tomodified expression of genes near the inserted T-DNA. The resultingtransgenic plants show dominant phenotypes due to modified expression ofgenes close to the introduced promoter.

TILLING

The term “TILLING” is an abbreviation of “Targeted Induced Local LesionsIn Genomes” and refers to a mutagenesis technology useful to generateand/or identify nucleic acids encoding proteins with modified expressionand/or activity. TILLING also allows selection of plants carrying suchmutant variants. These mutant variants may exhibit modified expression,either in strength or in location or in timing (if the mutations affectthe promoter for example). These mutant variants may exhibit higheractivity than that exhibited by the gene in its natural form. TILLINGcombines high-density mutagenesis with high-throughput screeningmethods. The steps typically followed in TILLING are: (a) EMSmutagenesis (Redei G P and Koncz C (1992) In Methods in ArabidopsisResearch, Koncz C, Chua N H, Schell J, eds. Singapore, World ScientificPublishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M,Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) InJ Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol.82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation andpooling of individuals; (c) PCR amplification of a region of interest;(d) denaturation and annealing to allow formation of heteroduplexes; (e)DHPLC, where the presence of a heteroduplex in a pool is detected as anextra peak in the chromatogram; (f) identification of the mutantindividual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (McCallum et al., (2000) NatBiotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet. 5(2):145-50).

Homologous Recombination

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offring a et al. (1990) EMBO J 9(10): 3077-84)but also for crop plants, for example rice (Terada et al. (2002) NatBiotech 20(10): 1030-4; Iida and Terada (2004) Curr Opin Biotech 15(2):132-8), and approaches exist that are generally applicable regardless ofthe target organism (Miller et al, Nature Biotechnol. 25, 778-785,2007).

Yield Related Traits

Yield related traits are traits or features which are related to plantyield. Yield-related traits may comprise one or more of the followingnon-limitative list of features: early flowering time, yield, biomass,seed yield, early vigour, greenness index, increased growth rate,improved agronomic traits, such as e.g. increased tolerance tosubmergence (which leads to increased yield in rice), improved Water UseEfficiency (WUE), improved Nitrogen Use Efficiency (NUE), etc.

Yield

The term “yield” in general means a measurable produce of economicvalue, typically related to a specified crop, to an area, and to aperiod of time. Individual plant parts directly contribute to yieldbased on their number, size and/or weight, or the actual yield is theyield per square meter for a crop and year, which is determined bydividing total production (includes both harvested and appraisedproduction) by planted square meters.

The terms “yield” of a plant and “plant yield” are used interchangeablyherein and are meant to refer to vegetative biomass such as root and/orshoot biomass, to reproductive organs, and/or to propagules such asseeds of that plant.

Flowers in maize are unisexual; male inflorescences (tassels) originatefrom the apical stem and female inflorescences (ears) arise fromaxillary bud apices. The female inflorescence produces pairs ofspikelets on the surface of a central axis (cob). Each of the femalespikelets encloses two fertile florets, one of them will usually matureinto a maize kernel once fertilized. Hence a yield increase in maize maybe manifested as one or more of the following: increase in the number ofplants established per square meter, an increase in the number of earsper plant, an increase in the number of rows, number of kernels per row,kernel weight, thousand kernel weight, ear length/diameter, increase inthe seed filling rate, which is the number of filled florets (i.e.florets containing seed) divided by the total number of florets andmultiplied by 100), among others.

Inflorescences in rice plants are named panicles. The panicle bearsspikelets, which are the basic units of the panicles, and which consistof a pedicel and a floret. The floret is borne on the pedicel andincludes a flower that is covered by two protective glumes: a largerglume (the lemma) and a shorter glume (the palea). Hence, taking rice asan example, a yield increase may manifest itself as an increase in oneor more of the following: number of plants per square meter, number ofpanicles per plant, panicle length, number of spikelets per panicle,number of flowers (or florets) per panicle; an increase in the seedfilling rate which is the number of filled florets (i.e. floretscontaining seeds) divided by the total number of florets and multipliedby 100; an increase in thousand kernel weight, among others.

Early Flowering Time

Plants having an “early flowering time” as used herein are plants whichstart to flower earlier than control plants. Hence this term refers toplants that show an earlier start of flowering. Flowering time of plantscan be assessed by counting the number of days (“time to flower”)between sowing and the emergence of a first inflorescence. The“flowering time” of a plant can for instance be determined using themethod as described in WO 2007/093444.

Early Vigour

“Early vigour” refers to active healthy well-balanced growth especiallyduring early stages of plant growth, and may result from increased plantfitness due to, for example, the plants being better adapted to theirenvironment (i.e. optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigour alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and often betterand higher yield. Therefore, early vigour may be determined by measuringvarious factors, such as thousand kernel weight, percentage germination,percentage emergence, seedling growth, seedling height, root length,root and shoot biomass and many more.

Increased Growth Rate

The increased growth rate may be specific to one or more parts of aplant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as speed of germination, early vigour,growth rate, greenness index, flowering time and speed of seedmaturation. The increase in growth rate may take place at one or morestages in the life cycle of a plant or during substantially the wholeplant life cycle. Increased growth rate during the early stages in thelife cycle of a plant may reflect enhanced vigour. The increase ingrowth rate may alter the harvest cycle of a plant allowing plants to besown later and/or harvested sooner than would otherwise be possible (asimilar effect may be obtained with earlier flowering time). If thegrowth rate is sufficiently increased, it may allow for the furthersowing of seeds of the same plant species (for example sowing andharvesting of rice plants followed by sowing and harvesting of furtherrice plants all within one conventional growing period). Similarly, ifthe growth rate is sufficiently increased, it may allow for the furthersowing of seeds of different plants species (for example the sowing andharvesting of corn plants followed by, for example, the sowing andoptional harvesting of soybean, potato or any other suitable plant).Harvesting additional times from the same rootstock in the case of somecrop plants may also be possible. Altering the harvest cycle of a plantmay lead to an increase in annual biomass production per square meter(due to an increase in the number of times (say in a year) that anyparticular plant may be grown and harvested). An increase in growth ratemay also allow for the cultivation of transgenic plants in a widergeographical area than their wild-type counterparts, since theterritorial limitations for growing a crop are often determined byadverse environmental conditions either at the time of planting (earlyseason) or at the time of harvesting (late season). Such adverseconditions may be avoided if the harvest cycle is shortened. The growthrate may be determined by deriving various parameters from growthcurves, such parameters may be: T-Mid (the time taken for plants toreach 50% of their maximal size) and T-90 (time taken for plants toreach 90% of their maximal size), amongst others.

Stress Resistance

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35%, 30% or 25%, more preferably less than 20% or 15% incomparison to the control plant under non-stress conditions. Due toadvances in agricultural practices (irrigation, fertilization, pesticidetreatments) severe stresses are not often encountered in cultivated cropplants. As a consequence, the compromised growth induced by mild stressis often an undesirable feature for agriculture. “Mild stresses” are theeveryday biotic and/or abiotic (environmental) stresses to which a plantis exposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures.

“Biotic stresses” are typically those stresses caused by pathogens, suchas bacteria, viruses, fungi, nematodes and insects.

The “abiotic stress” may be an osmotic stress caused by a water stress,e.g. due to drought, salt stress, or freezing stress. Abiotic stress mayalso be an oxidative stress or a cold stress. “Freezing stress” isintended to refer to stress due to freezing temperatures, i.e.temperatures at which available water molecules freeze and turn intoice. “Cold stress”, also called “chilling stress”, is intended to referto cold temperatures, e.g. temperatures below 10°, or preferably below5° C., but at which water molecules do not freeze. As reported in Wanget al. (Planta (2003) 218: 1-14), abiotic stress leads to a series ofmorphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signalling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location. Plants withoptimal growth conditions, (grown under non-stress conditions) typicallyyield in increasing order of preference at least 97%, 95%, 92%, 90%,87%, 85%, 83%, 80%, 77% or 75% of the average production of such plantin a given environment. Average production may be calculated on harvestand/or season basis. Persons skilled in the art are aware of averageyield productions of a crop.

In particular, the methods of the present invention may be performedunder non-stress conditions. In an example, the methods of the presentinvention may be performed under non-stress conditions such as milddrought to give plants having increased yield relative to controlplants.

In another embodiment, the methods of the present invention may beperformed under stress conditions.

In an example, the methods of the present invention may be performedunder stress conditions such as drought to give plants having increasedyield relative to control plants.

In another example, the methods of the present invention may beperformed under stress conditions such as nutrient deficiency to giveplants having increased yield relative to control plants.

Nutrient deficiency may result from a lack of nutrients such asnitrogen, phosphates and other phosphorous-containing compounds,potassium, calcium, magnesium, manganese, iron and boron, amongstothers.

In yet another example, the methods of the present invention may beperformed under stress conditions such as salt stress to give plantshaving increased yield relative to control plants. The term salt stressis not restricted to common salt (NaCl), but may be any one or more of:NaCl, KCl, LiCl, MgCl₂, CaCl₂, amongst others.

In yet another example, the methods of the present invention may beperformed under stress conditions such as cold stress or freezing stressto give plants having increased yield relative to control plants.

Increase/Improve/Enhance

The terms “increase”, “improve” or “enhance” are interchangeable andshall mean in the sense of the application at least a 3%, 4%, 5%, 6%,7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%,30%, 35% or 40% more yield and/or growth in comparison to control plantsas defined herein.

Seed Yield

Increased seed yield may manifest itself as one or more of thefollowing:

-   -   (a) an increase in seed biomass (total seed weight) which may be        on an individual seed basis and/or per plant and/or per square        meter;    -   (b) increased number of flowers per plant;    -   (c) increased number of seeds;    -   (d) increased seed filling rate (which is expressed as the ratio        between the number of filled florets divided by the total number        of florets);    -   (e) increased harvest index, which is expressed as a ratio of        the yield of harvestable parts, such as seeds, divided by the        biomass of aboveground plant parts; and    -   (f) increased thousand kernel weight (TKW), which is        extrapolated from the number of seeds counted and their total        weight. An increased TKW may result from an increased seed size        and/or seed weight, and may also result from an increase in        embryo and/or endosperm size.

The terms “filled florets” and “filled seeds” may be consideredsynonyms.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter.

Greenness Index

The “greenness index” as used herein is calculated from digital imagesof plants. For each pixel belonging to the plant object on the image,the ratio of the green value versus the red value (in the RGB model forencoding color) is calculated. The greenness index is expressed as thepercentage of pixels for which the green-to-red ratio exceeds a giventhreshold. Under normal growth conditions, under salt stress growthconditions, and under reduced nutrient availability growth conditions,the greenness index of plants is measured in the last imaging beforeflowering. In contrast, under drought stress growth conditions, thegreenness index of plants is measured in the first imaging afterdrought.

Biomass

The term “biomass” as used herein is intended to refer to the totalweight of a plant. Within the definition of biomass, a distinction maybe made between the biomass of one or more parts of a plant, which mayinclude any one or more of the following:

-   -   aboveground parts such as but not limited to shoot biomass, seed        biomass, leaf biomass, etc.;    -   aboveground harvestable parts such as but not limited to shoot        biomass, seed biomass, leaf biomass, etc.;    -   parts below ground, such as but not limited to root biomass,        tubers, bulbs, etc.;    -   harvestable parts below ground, such as but not limited to root        biomass, tubers, bulbs, etc.;    -   harvestable parts partially below ground such as but not limited        to beets and other hypocotyl areas of a plant, rhizomes, stolons        or creeping rootstalks;    -   vegetative biomass such as root biomass, shoot biomass, etc.;    -   reproductive organs; and    -   propagules such as seed.

Marker Assisted Breeding

Such breeding programmes sometimes require introduction of allelicvariation by mutagenic treatment of the plants, using for example EMSmutagenesis; alternatively, the programme may start with a collection ofallelic variants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

Use as Probes in (Gene Mapping)

Use of nucleic acids encoding the protein of interest for geneticallyand physically mapping the genes requires only a nucleic acid sequenceof at least 15 nucleotides in length. These nucleic acids may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blots(Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, ALaboratory Manual) of restriction-digested plant genomic DNA may beprobed with the nucleic acids encoding the protein of interest. Theresulting banding patterns may then be subjected to genetic analysesusing computer programs such as MapMaker (Lander et al. (1987) Genomics1: 174-181) in order to construct a genetic map. In addition, thenucleic acids may be used to probe Southern blots containing restrictionendonuclease-treated genomic DNAs of a set of individuals representingparent and progeny of a defined genetic cross. Segregation of the DNApolymorphisms is noted and used to calculate the position of the nucleicacid encoding the protein of interest in the genetic map previouslyobtained using this population (Botstein et al. (1980) Am. J. Hum.Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

Plant

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubsselected from the list comprising Acer spp., Actinidia spp., Abelmoschusspp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef,Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora,Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g.Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthusspp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp.,Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp.,Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum,Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzulasylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp.,Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp.,Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotianaspp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryzasativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum,Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp.,Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleumpratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp.,Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunusspp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.,Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubusspp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamumspp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanumintegrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticumspp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum,Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcumor Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vacciniumspp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays,Zizania palustris, Ziziphus spp., amongst others.

Control Plant(s)

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. Nullizygotes (or null control plants) areindividuals missing the transgene by segregation. Further, controlplants are grown under equal growing conditions to the growingconditions of the plants of the invention, i.e. in the vicinity of, andsimultaneously with, the plants of the invention. A “control plant” asused herein refers not only to whole plants, but also to plant parts,including seeds and seed parts.

DETAILED DESCRIPTION OF THE INVENTION

Concerning DEAD-box RNA helicase polypeptides, it has now beensurprisingly found that reducing or substantially eliminating expressionin a plant of a nucleic acid encoding a DEAD-box RNA helicasepolypeptide gives plants having enhanced yield-related traits relativeto control plants.

Concerning CER2-like polypeptides, it has now been surprisingly foundthat modulating expression in a plant of a nucleic acid encoding aCER2-like polypeptide gives plants having enhanced yield-related traitsrelative to control plants.

Concerning At1g68440-like polypeptides, it has now been found thatmodulating expression in a plant of a nucleic acid encoding anAt1g68440-like polypeptide gives plants having enhanced yield-relatedtraits relative to control plants.

Concerning DEAD-box RNA helicase polypeptides, according to a firstembodiment, the present invention provides a method for enhancingyield-related traits in plants relative to control plants, comprisingreducing or substantially eliminating expression in a plant of a nucleicacid encoding an endogenous DEAD-box RNA helicase polypeptide andoptionally selecting for plants having enhanced yield-related traits.

According to another embodiment, the present invention provides a methodfor producing plants having enhanced yield-related traits relative tocontrol plants, wherein said method comprises the steps of reducing orsubstantially eliminating expression in said plant of a nucleic acidencoding an endogenous DEAD-box RNA helicase polypeptide as describedherein and optionally selecting for plants having enhanced yield-relatedtraits.

In a preferred embodiment, the reduction or substantial elimination ofexpression of an endogenous DEAD-box RNA helicase gene and/or leveland/or activity of an endogenous DEAD-box RNA helicase protein isobtained by introducing a DEAD-box RNA helicase nucleic acid or fragmentthereof substantially homologous to said endogenous DEAD-box RNAhelicase gene, more preferably said isolated nucleic acid is capable offorming a hairpin structure, further preferably the isolated nucleicacid is under the control of a constitutive promoter.

Concerning CER2-like polypeptides, according to a first embodiment, thepresent invention provides a method for enhancing yield-related traitsin plants relative to control plants, comprising modulating expressionin a plant of a nucleic acid encoding a CER2-like polypeptide andoptionally selecting for plants having enhanced yield-related traits.Preferably, a method is provided for enhancing yield-related traits inplants relative to control plants, comprising modulating expression in aplant of a nucleic acid encoding a CER2-like polypeptide or a homologuethereof, wherein said CER2-like polypeptide or homologue thereofcomprises a CER2-like related domain.

A preferred method for modulating, and preferably for increasingexpression of a nucleic acid encoding a CER2-like polypeptide asprovided herein or a homologue thereof is by introducing and expressingin a plant a nucleic acid encoding a CER2-like polypeptide or saidhomologue.

In an embodiment, a method is provided wherein said enhancedyield-related traits comprise increased yield relative to controlplants, and preferably comprise increased seed yield and/or increasedbiomass relative to control plants.

In one embodiment a method is provided wherein said enhancedyield-related traits are obtained under non-stress conditions.

In another embodiment, a method is provided wherein said enhancedyield-related traits are obtained under conditions of osmotic stress,such as for instance drought stress, cold stress and/or salt stress, orunder conditions of nitrogen deficiency.

Concerning At1g68440-like polypeptides, according to a first embodiment,the present invention provides a method for enhancing yield-relatedtraits in plants relative to control plants, comprising modulatingexpression in a plant of a nucleic acid encoding an At1g68440-likepolypeptide and optionally selecting for plants having enhancedyield-related traits.

A preferred method for modulating, preferably increasing, expression ofa nucleic acid encoding an At1g68440-like polypeptide is by introducingand expressing in a plant a nucleic acid encoding an At1g68440-likepolypeptide.

In one embodiment a “protein useful in the methods of the invention” istaken to mean a DEAD-box RNA helicase polypeptide as defined herein. Anyreference hereinafter to a “nucleic acid useful in the methods of theinvention” is taken to mean a nucleic acid capable of encoding such aDEAD-box RNA helicase polypeptide. The nucleic acid to be introducedinto a plant (and therefore useful in performing the methods of theinvention) is any nucleic acid encoding the type of protein which willnow be described, hereafter also named “DEAD-box RNA helicase nucleicacid” or “DEAD-box RNA helicase gene”.

A “DEAD-box RNA helicase polypeptide” as defined herein refers to anypolypeptide comprising signature pattern[LIVMF]-[LIVMF]-D-E-A-D-[RKEN]-X-[LIVMFYGSTN] (SEQ ID NO: 159).

Aubourg et al. (1999) proposed a relationship tree of 25 A. thalianaDEAD-box RNA helicase polypeptides (see FIG. 6). When analysed in thatphylogenetic tree, DEAD-box RNA helicases tend to cluster together in 6distinct subfamilies.

In a preferred embodiment, the DEAD-box RNA helicase polypeptide usefulin the methods of the present invention, is an orthologue or paralogueto subfamily VI of the 6 subfamilies of DEAD-box RNA helicases asdescribed by Aubourg et al. (1999).

In a more preferred embodiment, the DEAD-box RNA helicase polypeptidecomprises one or more of the following MEME motifs:

(SEQ ID NO: 3) (i) Motif 1: [LM][VI]ATDVA[AS]RGLD[IV][KP]D[VI][EK]VV[IV]N[YF][SD][YF]P[LN][TD][IT] [ED]DYVHRIGRTGRA, (SEQ ID NO: 4)(ii) Motif 2: [RSN]L[NR][DR]V[TS][YF][LV]VLDEADRMLDMGFEP[EQ][IV]R[AK]I[VL], (SEQ ID NO: 5) (iii) Motif 3:P[TS]PIQA[YQ][AS][WI]P[YI][AL][LM] [DS]GRD[FL][IV][GA]IA[KA]TGSGKT

In an even more preferred embodiment, the DEAD-box RNA helicasepolypeptide comprises one or more of the following MEME motifs:

(SEQ ID NO: 6) (i) Motif 4: ATDVA[SA]RGLD[IV][KP]D[VI][EKR][VY]V[IV]NY[DS][YF]P[LT][TG][TLV]EDYVHRIG RTGR[TA]G[RAK][KT]G[VLT]A[HY]TFFT(SEQ ID NO: 7) (ii) Motif 5: [MS][GR][IV][CIT][RNS]L[NR][DRQE]V[ST][YF][LV]VLDEADRMLDMGFEP[QE][IV] R[KA]I[VL][SG][QE][TI][ARP][PS][VD]RQ[TM][LV]M[FWY][ST]ATWP (SEQ ID NO: 8) (iii) Motif 6:GF[ES][REK]P[ST]PIQ[AS][YQ][SAG]WP [YIMF][AL][LM][DKQ]GRD[FLI][IV][GA]IA [AKE]TGSGKT[IL][AG][FY][LG][VLI] P[AG][LFI][MV]H[VIL]

Even more preferably, the DEAD-box RNA helicase polypeptide comprisesone or more of the following MEME motifs:

(SEQ ID NO: 9) (i) Motif 7: [IV]ATDVA[AS]RGLDIPDVE[VY]VINY[ST][YF]PLTTEDYVHRIGRTGRAGK[KT]G[VL]AH TFF (SEQ ID NO: 10) (ii) Motif 8:VLDEADRMLD[ML]GFEPE[VI]R[AS]I[LA] [GS]QT[ACR][SA][DV]RQ[MT]VMFSATWP[PL][AS]V[HQ][KQ]LAQEFMD (SEQ ID NO: 11) (iii) Motif 9:VL[DE]C[CT]KGF[EQ][KR]PSPIQ[AS] [YHQ]AWP[YFI]LL[DS]GRD[FL][IV]GI AATGSGKT[LI]AFG[VI]PALMH[VI]

Most preferably, the DEAD-box RNA helicase polypeptide comprises inincreasing order of preference 1, 2, 3, 4, 5, 6, 7, or all of the motifs10 to 17:

(SEQ ID NO: 160) (i) motif 10: FXXPSPIQ (SEQ ID NO: 161) (ii) motif 11:AXTGSGKT (SEQ ID NO: 162) (iii) motif 12: PTRELAXQ (SEQ ID NO: 163) (iv)motif 13: TPGR (SEQ ID NO: 164) (v) motif 14: VLDEADRMLD(SEQ ID NO: 165) (vi) motif 15: MFSATWP (SEQ ID NO: 166) (vii) motif 16:ATDVA[AS]RGLDI (SEQ ID NO: 167) (viii) motif 17: RIGRTGRAG

In an alternative more preferred embodiment, the DEAD-box RNA helicasepolypeptide comprises one motif from the group of motifs 1, 4 and 7and/or one motif of the group of motifs 2, 5 and 8 and/or one motif ofthe group of motifs 3, 6 and 9.

Motifs 1 to 9 were derived using the MEME algorithm (Bailey and Elkan,Proceedings of the Second International Conference on IntelligentSystems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park,Calif., 1994). At each position within a MEME motif, the residues areshown that are present in the query set of sequences with a frequencyhigher than 0.2. Residues within square brackets represent alternatives.

Motifs 10 to 17 were derived from the multiple alignment as shown inFIG. 2. Motifs 10 to 17 are highly conserved motifs in the DEAD-box RNAhelicase polypeptides. From the alignment of FIG. 2 more conservedregions can be deduced.

Additionally or alternatively, the homologue of a DEAD-box RNA helicaseprotein has in increasing order of preference at least 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% overall sequence identity to the amino acid represented bySEQ ID NO: 2, provided that the homologous protein comprises any one ormore of the conserved motifs as outlined above. The overall sequenceidentity is determined using a global alignment algorithm, such as theNeedleman Wunsch algorithm in the program GAP (GCG Wisconsin Package,Accelrys), preferably with default parameters and preferably withsequences of mature proteins (i.e. without taking into account secretionsignals or transit peptides). Compared to overall sequence identity, thesequence identity will generally be higher when only conserved domainsor motifs are considered. Preferably the motifs in a DEAD-box RNAhelicase polypeptide have, in increasing order of preference, at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to any one or more of the motifsrepresented by SEQ ID NO: 3 to SEQ ID NO: 11. More preferably the motifsin a DEAD-box RNA helicase polypeptide have, in increasing order ofpreference, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one or more ofthe motifs represented by SEQ ID NO: 160 to SEQ ID NO: 167.

In another embodiment a “protein useful in the methods of the invention”is taken to mean a CER2-like polypeptide as defined herein. Anyreference hereinafter to a “nucleic acid useful in the methods of theinvention” is taken to mean a nucleic acid capable of encoding such aCER2-like polypeptide. The nucleic acid to be introduced into a plant(and therefore useful in performing the methods of the invention) is anynucleic acid encoding the type of protein which will now be described,hereafter also named “CER2-like nucleic acid” or “CER2-like gene”.

A “CER2-like polypeptide” as defined herein refers to any Pfam PF02458domain containing protein or polypeptide belonging to acyltransferasefamily. These proteins are involved in multiple biosynthetic pathwaysincluding, but not limited to, phytoalexin, vindoline and cuticular waxbiosynthesis as well as detoxification of toxic metabolites. Saidacyltransferase falls in two main categories:

-   -   Clade A members, including Arabidopsis CER2 and corn glossy 2,        which are potentially involved in wax biosynthesis (see FIG. 9),    -   DAT-like acyl transferase represented by Glade B members (see        FIG. 9) and characterized by a conserved D[FL]G[GW]Gx[PE] motif.

Enzymes having said activity, for example, include: anthranilateN-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline4-O-acetyltransferase (DAT, EC:2.3.1.107) involved in the last step invindoline biosynthesis. More particular, this subfamily is characterizedby a HxxxDG triad and a DFGWGKP consensus sequence. Also included inthis family is, for example, Trichothecene 3-O-acetyltransferase whichis involved in the detoxification of trichothecene. These polypeptideshave been previously described to be involved in cuticular waxbiosynthesis in plants, as described supra. The term “CER2-likepolypeptide” as used herein also intends to include homologues asdefined hereunder of “CER2-like polypeptides”.

In a preferred embodiment, a CER2-like polypeptide as applied hereincomprises a CER2-like related domain. In a preferred embodiment, theCER2-like related domain corresponds to Pfam PF02458.

In a preferred embodiment, the CER2-like polypeptide comprises one ormore of the following motifs:

i) Motif 18a: (SEQ ID NO: 274)FKCGGL[SA][LI]G[LV][SG]W[AS]HI[LV][GA]D[GA]F SASHFIN[SA]W.Preferably said motif is (motif 18b; SEQ ID NO: 275)FKCGGLSVGLSWAHILGDAFSAFNFITKWSHILAGQSQPKS ii) Motif 19a:(SEQ ID NO: 276) SRG[PL]E[IVL]KCNDEG[VA][RL][FI][VI]EAE[CA]D.Pregerably, said motif is (motif 19b; SEQ ID NO: 277)SGRPFIKCNDAGVRIAESQCDK; iii) Motif 20a: (SEQ ID NO: 278)xY[ST][TR][FY]E[AI]L[AST][AG][HL][IV]W[RK] [CS]I[AC]KARG.Preferably, said motif is (motif 20b; SEQ ID NO: 279)DTKYFEIISATIWKCIAQIRG.

In one preferred embodiment, the CER2-like polypeptide comprises alsoone or more of the following motifs:

i) Motif 21a: (SEQ ID NO: 280)VQ[VF]TxFKCGG[LM][SA][LIV]GLS[WC]AH[IVL]LGD [APV]FSA[ST]TF[FIM][NK]KW.Preferably said motif is: (motif 21b; SEQ ID NO: 281)VFVKFTSFKCGGLSVGLSWAHILGDAFSAFNFITKW; ii) Motif 22a: (SEQ ID NO: 282)[EA]SGR[PW][YF][IV]KCND[AC]GVRIVEA[KHR]CDK. Preferably said motif is:(motif 22b; SEQ ID NO: 283) KSGFPFIKCNDAGVRIAESQCDK; iii) Motif 23a:(SEQ ID NO: 284) SR[VT][GE][EP][GN]Kx[HY]E[LP][ST]x[LM]DLAMKLHY[LVI]R[GA]VY[FY][FY]. Preferably said motif is:(motif 23b; SEQ ID NO: 285) GEHNLNYMDLLMKLHY;

These motifs 18 to 23 are essentially present in CER2-like polypeptidesof the Clade A group of polypeptides as described herein.

In yet another preferred embodiment, the CER2-like polypeptide comprisesalso one or more of the following motifs:

i) Motif 24: (SEQ ID NO: 286)FKCGG[VIF][SA][LI]G[VL]G[MI]SHx[VLM]ADGxSALHF [NS][SAT]W. ii) Motif 25:(SEQ ID NO: 287) NPNLL[IV][TV]SWT[RT][LF]P[ILF][YH][DE]ADFGWG[KR]PI[FY]MGP; iii) Motif 26: (SEQ ID NO: 288)[SA]LS[KE][VT]L[VT][HP][FY]YP[ML]AGR;

These additional motifs 7 to 9 are essentially present in CER2-likepolypeptides of the DAT-like acyl transferase represented by Glade Bmembers as described herein.

As used herein, the letter “x” means any amino acid and [AB] meanseither A or B amino acid is possible at this position.

Motifs 18 to 26 were derived using the MEME algorithm (Bailey and Elkan,Proceedings of the Second International Conference on IntelligentSystems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park,Calif., 1994). At each position within a MEME motif, the residues areshown that are present in the query set of sequences with a frequencyhigher than 0.2. Residues within square brackets represent alternatives.

More preferably, the CER2-like polypeptide comprises in increasing orderof preference, at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, or all 9 motifs selected from the groupcomprising motifs 18a, 19a, 20a, 21a, 22a, 23a, 24, 25, and 26. In aneven more preferred embodiment, the CER2-like polypeptide comprises atleast 2, at least 3, at least 4, at least 5, or all 6 motifs selectedfrom the group comprising motifs 18a, 19a, 20a, 21a, 22a, 23a. In aparticularly preferred embodiment, the CER2-like polypeptide comprisesat least 2, at least 3, at least 4, at least 5, or all 6 motifs selectedfrom the group comprising motifs 18b, 19b, 20b, 21b, 22b, 23b.

Additionally or alternatively, the homologue of a CER2-like protein hasin increasing order of preference at least 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% overall sequence identity to the amino acidrepresented by SEQ ID NO: 169, provided that the homologous proteincomprises any one or more of the conserved Motifs 18 to 26 as outlinedabove. The overall sequence identity is determined using a globalalignment algorithm, such as the Needleman Wunsch algorithm in theprogram GAP (GCG Wisconsin Package, Accelrys), preferably with defaultparameters and preferably with sequences of mature proteins (i.e.without taking into account secretion signals or transit peptides).Compared to overall sequence identity, the sequence identity willgenerally be higher when only conserved domains or motifs areconsidered. Preferably the motifs in a CER2-like polypeptide have, inincreasing order of preference, at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toany one or more of the motifs represented by SEQ ID NO: 274 to SEQ IDNO: 285 (Motifs 18 to 23).

In other words, in another embodiment a method is provided wherein saidCER2-like polypeptide comprises a conserved domain (or motif) with atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to a conserved domain of amino acidcoordinates 73 to 189 of SEQ ID NO: 169).

Any reference hereinafter to a “protein useful in the methods of theinvention” is taken to mean an At1g68440-like polypeptide as definedherein. Any reference hereinafter to a “nucleic acid useful in themethods of the invention” is taken to mean a nucleic acid capable ofencoding such an At1g68440-like polypeptide. The nucleic acid to beintroduced into a plant (and therefore useful in performing the methodsof the invention) is any nucleic acid encoding the type of protein whichwill now be described, hereafter also named

“At1g68440-like nucleic acid” or “At1g68440-like gene”.

A “At1g68440-like polypeptide” preferably comprises a transmembranedomain.

The terms “domain”, “signature”, and “motif” are defined in thedefinitions section herein.

In a preferred embodiment, the At1g68440-like polypeptide comprises oneor more of the following motifs:

i) Motif 27a: (SEQ ID NO: 364) Y[SN]F[WC][KT]WGALILA[LV][FVL]A.Preferably said motif is (motif 26b; SEQ ID NO: 365) YSFWKWGALILALAA.ii) Motif 28a: (SEQ ID NO: 366) ME[IV][PT][VE][IL]N[RL]I[SG]DF.Preferably, said motif is (motif 28b; SEQ ID NO: 367) MEIPVINRISDF; iii)Motif 29a: (SEQ ID NO: 368) [SN]VV[KQ]LWD[SN]LG[LF].Preferably, said motif is (motif 29b; SEQ ID NO: 369) NVVKLWDNLGL.

In one preferred embodiment, the At1g68440-like polypeptide comprisesalso one or more of the following motifs:

i) Motif 30a: (SEQ ID NO: 370) SQ[VI][SL]A[LV]SG[IV][GE]K[FI][YH]Q[AT]Y[NSH][FL]WKWGALILAL[IAV]ASF[TS]IINR[VI]K [AI]L[IV][KR][LF][QK][KN]HP.Preferably said motif is: (motif 30b; SEQ ID NO: 371)SQILALSGVEKIHQAYSFWKWGALILALAASFTAIINRIK LIIRFKNHP; ii) Motif 31a:(SEQ ID NO: 372) [MDE][MF]T[SN]GK[NS]VVKLWDNLGLG[LF]G.Preferably said motif is: (motif 31b; SEQ ID NO: 373)DFTNGKNVVKLWDNLGLSLGL; iii) Motif 32a: (SEQ ID NO: 374)MEI[LP]V[IV]NRI[IS]DFE[TA][NGS][IL][NAS] [PS][SL][LQ][VN].Preferably said motif is: (motif 32b; SEQ ID NO: 375)MEIPVINRISDFETGLASLQN;

These motifs 27 to 32 are essentially present in At1g68440-likepolypeptides of the Clade A group of polypeptides as described herein.

As used herein, the letter “x” means any amino acid and [AB] meanseither A or B amino acid is possible at this position.

Motifs 1 to 6 were derived using the MEME algorithm (Bailey and Elkan,Proceedings of the Second International Conference on IntelligentSystems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park,Calif., 1994). At each position within a MEME motif, the residues areshown that are present in the query set of sequences with a frequencyhigher than 0.2. Residues within square brackets represent alternatives.

More preferably, the At1g68440-like polypeptide comprises in increasingorder of preference, at least 2, at least 3, at least 4, at least 5, orall 6 motifs, selected from the group comprising motifs 27a, 28a, 29a,30a, 31a, and 32a. In a particularly preferred embodiment, theAt1g68440-like polypeptide comprises at least 2, at least 3, at least 4,at least 5, or all 6 motifs selected from the group comprising motifs27b, 28b, 29b, 30b, 31b, and 32b.

The term “At1g68440-like polypeptide” as used herein also intends toinclude homologues as defined hereunder of “At1g68440-like polypeptide”.

Additionally or alternatively, the homologue of an At1g68440-likeprotein has in increasing order of preference at least 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% overall sequence identity to the amino acid represented bySEQ ID NO: 292, provided that the homologous protein comprises any oneor more of the conserved motifs 27 to 32 as outlined above. The overallsequence identity is determined using a global alignment algorithm, suchas the Needleman Wunsch algorithm in the program GAP (GCG WisconsinPackage, Accelrys), preferably with default parameters and preferablywith sequences of mature proteins (i.e. without taking into accountsecretion signals or transit peptides). Compared to overall sequenceidentity, the sequence identity will generally be higher when onlyconserved domains or motifs are considered. Preferably the motifs in anAt1g68440-like polypeptide have, in increasing order of preference, atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any one or more of the motifsrepresented by SEQ ID NO: 370 to SEQ ID NO: 375 (Motifs 27 to 32).

In other words, in another embodiment a method is provided wherein saidAt1g68440-like polypeptide comprises a transmembrane domain with atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to a conserved domain of amino acidcoordinates 46 to 64 of SEQ ID NO: 292.

The terms “domain”, “signature” and “motif” are defined in the“definitions” section herein.

Concerning DEAD-box RNA helicase polypeptides, preferably, thepolypeptide sequence which when used in the construction of aphylogenetic tree, such as the one depicted in FIG. 3, clusters with thegroup of DEAD-box RNA helicase polypeptides (preferably of subgroup VIas defined by Aubourg et al. (1999)) comprising the amino acid sequencerepresented by SEQ ID NO: 2 rather than with any other group.

Furthermore, DEAD-box RNA helicase polypeptides (at least in theirnative form) typically have RNA unwinding activity. Tools and techniquesfor measuring helicase activity are well known in the art, e.g. Okanamiet al. (1998) Nucleic Acid Res. (26): 2638-2643.

In addition, DEAD-box RNA helicase polypeptides, when reduced orsubstantially eliminated in transgenic plants such as e.g. riceaccording to the methods of the present invention as outlined in TheExamples section, give plants having increased yield related traits, inparticular increased seed yield.

Concerning CER2-like polypeptides, preferably, the polypeptide sequencewhich when used in the construction of a phylogenetic tree, such as theone depicted in FIG. 9, clusters with the group of CER2-likepolypeptides of Clade A comprising the amino acid sequence representedby SEQ ID NO: 167 rather than with any other group.

Furthermore, CER2-like polypeptides (at least in their native form)typically have acyltransferase activity. Tools and techniques formeasuring acyltransferase activity are well known in the art.Preferably, the CER2-like polypeptide belongs to EC 2.3.1. Depending onthe type of acyltransferase, assays are available in the art to measuresuch an activity. As an example, Power et al., 1985, (Archives ofBiochemistry and Biophysics, Vol. 279, pages 370-376) describes howDeacetylvindoline 4-O-acetyltransferase activity can be determined.Further details are provided in the Examples section.

In addition, CER2-like polypeptides, when expressed in rice according tothe methods of the present invention as outlined in the Examplessection, give plants having increased yield related traits, inparticular increased total weight of seeds, increased seed fill rate,increased harvest index, increased number of filled seeds.

Concerning At1g68440-like polypeptides, preferably, the polypeptidesequence which when used in the construction of a phylogenetic tree,such as the one depicted in FIG. 14, clusters with the group ofAt1g68440-like polypeptides of Clade A comprising the amino acidsequence represented by SEQ ID NO: 292 rather than with any other group.

In addition, At1g68440-like polypeptides, when expressed in riceaccording to the methods of the present invention as outlined in theExamples section, give plants having increased yield related traits, inparticular increased seed yield, number of filled seeds, seed fillrate,total seed weight or HarvestIndex.

Concerning DEAD-box RNA helicase polypeptides, the present invention isillustrated by transforming plants with the nucleic acid sequencerepresented by SEQ ID NO: 1, encoding the polypeptide sequence of SEQ IDNO: 2. However, performance of the invention is not restricted to thesesequences; the methods of the invention may advantageously be performedusing any DEAD-box RNA helicase-encoding nucleic acid or DEAD-box RNAhelicase polypeptide as defined herein, provided this DEAD-box RNAhelicase gene reduces or substantially eliminates expression of theendogenous DEAD-box RNA helicase polypeptide.

Examples of nucleic acids encoding DEAD-box RNA helicase polypeptidesare given in Table A1 of the Examples section herein. Such nucleic acidsare useful in performing the methods of the invention. The amino acidsequences given in Table A1 of the Examples section are examplesequences of orthologues and paralogues of the DEAD-box RNA helicasepolypeptide represented by SEQ ID NO: 2, the terms “orthologues” and“paralogues” being as defined herein. Further orthologues and paraloguesmay readily be identified by performing a so-called reciprocal blastsearch as described in the definitions section; where the query sequenceis SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST (back-BLAST) would beagainst rice sequences.

The invention also provides hitherto unknown DEAD-box RNAhelicase-encoding nucleic acids and DEAD-box RNA helicase polypeptidesuseful for conferring enhanced yield-related traits in plants relativeto control plants.

According to a further embodiment of the present invention, there istherefore provided an isolated nucleic acid molecule selected from:

-   -   (i) a nucleic acid represented by SEQ ID NO: 12 or 14;    -   (ii) the complement of a nucleic acid represented by SEQ ID NO:        12 or 14;    -   (iii) a nucleic acid encoding a DEAD-box RNA helicase        polypeptide having in increasing order of preference at least        50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,        63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,        76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,        89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%        sequence identity to the amino acid sequence represented by SEQ        ID NO: 13 or 15, and additionally or alternatively comprising        one or more motifs having in increasing order of preference at        least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,        97%, 98%, 99% or more sequence identity to any one or more of        the motifs given in SEQ ID NO: 3 to SEQ ID NO: 11, and further        preferably conferring enhanced yield-related traits relative to        control plants.    -   (iv) a nucleic acid molecule which hybridizes with a nucleic        acid molecule of (i) to (iii) under high stringency        hybridization conditions and preferably confers enhanced        yield-related traits relative to control plants.

According to a further embodiment of the present invention, there isalso provided an isolated polypeptide selected from:

-   -   (i) an amino acid sequence represented by SEQ ID NO: 13 or 15;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by SEQ ID NO: 13 or 15, and additionally or        alternatively comprising one or more motifs having in increasing        order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to        any one or more of the motifs given in SEQ ID NO: 3 to SEQ ID        NO: 11, and further preferably conferring enhanced yield-related        traits relative to control plants;    -   (iii) derivatives of any of the amino acid sequences given        in (i) or (ii) above.

Concerning CER2-like polypeptides, the present invention is illustratedby transforming plants with the nucleic acid sequence represented by SEQID NO: 168, encoding the polypeptide sequence of SEQ ID NO: 169.However, performance of the invention is not restricted to thesesequences; the methods of the invention may advantageously be performedusing any CER2-like-encoding nucleic acid or CER2-like polypeptide asdefined herein.

Examples of nucleic acids encoding CER2-like polypeptides are given inTable A2 of the Examples section herein. Such nucleic acids are usefulin performing the methods of the invention. The amino acid sequencesgiven in Table A2 of the Examples section are example sequences oforthologues and paralogues of the CER2-like polypeptide represented bySEQ ID NO: 169, the terms “orthologues” and “paralogues” being asdefined herein. Further orthologues and paralogues may readily beidentified by performing a so-called reciprocal blast search asdescribed in the definitions section; where the query sequence is SEQ IDNO: 168 or SEQ ID NO: 169, the second BLAST (back-BLAST) would beagainst alfalfa sequences.

The invention also provides hitherto unknown CER2-like-encoding nucleicacids and CER2-like polypeptides useful for conferring enhancedyield-related traits in plants relative to control plants.

According to a further embodiment of the present invention, there istherefore provided an isolated nucleic acid molecule selected from:

-   -   (i) a nucleic acid represented by SEQ ID NO: 168;    -   (ii) the complement of a nucleic acid represented by SEQ ID NO:        168;    -   (iii) a nucleic acid encoding a CER2-like polypeptide having in        increasing order of preference at least 50%, 51%, 52%, 53%, 54%,        55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,        68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,        81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino        acid sequence represented by SEQ ID NO: 169 and additionally or        alternatively comprising one or more motifs having in increasing        order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to        any one or more of the motifs given in SEQ ID NO: 274 to SEQ ID        NO: 288, and further preferably conferring enhanced        yield-related traits relative to control plants.    -   (iv) a nucleic acid molecule which hybridizes with a nucleic        acid molecule of (i) to (iii) under high stringency        hybridization conditions and preferably confers enhanced        yield-related traits relative to control plants.    -   (v) a nucleic acid encoding a CER2-like polypeptide having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by SEQ ID NO: 169 and any of the        other amino acid sequences in Table A2 and preferably conferring        enhanced yield-related traits relative to control plants.

According to a further embodiment of the present invention, there isalso provided an isolated polypeptide selected from:

-   -   (i) an amino acid sequence represented by SEQ ID NO: 169;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by SEQ ID NO: 169, and additionally or alternatively        comprising one or more motifs having in increasing order of        preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, 99% or more sequence identity to any one or        more of the motifs given in SEQ ID NO: 274 to SEQ ID NO: 288,        and further preferably conferring enhanced yield-related traits        relative to control plants;    -   (iii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by (any one of) SEQ ID NO: 169 and any of the other        amino acid sequences in Table A2 and preferably conferring        enhanced yield-related traits relative to control plants;    -   (iv) derivatives of any of the amino acid sequences given in (i)        or (iii) above.

Concerning At1g68440-like polypeptides, the present invention isillustrated by transforming plants with the nucleic acid sequencerepresented by SEQ ID NO: 291, encoding the polypeptide sequence of SEQID NO: 292. However, performance of the invention is not restricted tothese sequences; the methods of the invention may advantageously beperformed using any At1g68440-like-encoding nucleic acid orAt1g68440-like polypeptide as defined herein.

Examples of nucleic acids encoding At1g68440-like polypeptides are givenin Table A3 of the Examples section herein. Such nucleic acids areuseful in performing the methods of the invention. The amino acidsequences given in Table A3 of the Examples section are examplesequences of orthologues and paralogues of the At1g68440-likepolypeptide represented by SEQ ID NO: 292, the terms “orthologues” and“paralogues” being as defined herein. Further orthologues and paraloguesmay readily be identified by performing a so-called reciprocal blastsearch as described in the definitions section; where the query sequenceis SEQ ID NO: 291 or SEQ ID NO: 292, the second BLAST (back-BLAST) wouldbe against poplar sequences.

The invention also provides hitherto unknown At1g68440-like-encodingnucleic acids and At1g68440-like polypeptides useful for conferringenhanced yield-related traits in plants relative to control plants.

According to a further embodiment of the present invention, there istherefore provided an isolated nucleic acid molecule selected from:

-   -   (i) a nucleic acid represented by SEQ ID NO: 291;    -   (ii) the complement of a nucleic acid represented by SEQ ID NO:        291;    -   (iii) a nucleic acid encoding an At1g68440-like polypeptide        having in increasing order of preference at least 50%, 51%, 52%,        53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,        66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,        79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to        the amino acid sequence represented by SEQ ID NO: 292, and        additionally or alternatively comprising one or more motifs        having in increasing order of preference at least 50%, 55%, 60%,        65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more        sequence identity to any one or more of the motifs given in SEQ        ID NO: 364 to SEQ ID NO: 375, and further preferably conferring        enhanced yield-related traits relative to control plants.    -   (iv) a nucleic acid molecule which hybridizes with a nucleic        acid molecule of (i) to (iii) under high stringency        hybridization conditions and preferably confers enhanced        yield-related traits relative to control plants.    -   (v) a nucleic acid encoding an At1g68440-like polypeptide        having, in increasing order of preference, at least 50%, 51%,        52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,        65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,        78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity        to the amino acid sequence represented by SEQ ID NO: 292 and any        of the other amino acid sequences in Table A3 and preferably        conferring enhanced yield-related traits relative to control        plants.

According to a further embodiment of the present invention, there isalso provided an isolated polypeptide selected from:

-   -   (i) an amino acid sequence represented by SEQ ID NO: 292;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by SEQ ID NO: 2, and additionally or alternatively        comprising one or more motifs having in increasing order of        preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, 99% or more sequence identity to any one or        more of the motifs given in SEQ ID NO: 364 to SEQ ID NO: 375,        and/or any of the other amino acid sequences in Table A3, and        further preferably conferring enhanced yield-related traits        relative to control plants;    -   (iii) derivatives of any of the amino acid sequences given        in (i) or (ii) above.

Nucleic acid variants may also be useful in practising the methods ofthe invention. Examples of such variants include nucleic acids encodinghomologues and derivatives of any one of the amino acid sequences givenin Table A1 to A3 of the Examples section, the terms “homologue” and“derivative” being as defined herein. Also useful in the methods of theinvention are nucleic acids encoding homologues and derivatives oforthologues or paralogues of any one of the amino acid sequences givenin Table A1 to A3 of the Examples section. Homologues and derivativesuseful in the methods of the present invention have substantially thesame biological and functional activity as the unmodified protein fromwhich they are derived. Further variants useful in practising themethods of the invention are variants in which codon usage is optimisedor in which miRNA target sites are removed.

Further nucleic acid variants useful in practising the methods of theinvention include portions of nucleic acids encoding DEAD-box RNAhelicase polypeptides, or CER2-like polypeptides, or At1g68440-likepolypeptides, nucleic acids hybridising to nucleic acids encodingDEAD-box RNA helicase polypeptides, or CER2-like polypeptides, orAt1g68440-like polypeptides, splice variants of nucleic acids encodingDEAD-box RNA helicase polypeptides, or CER2-like polypeptides, orAt1g68440-like polypeptides, allelic variants of nucleic acids encodingDEAD-box RNA helicase polypeptides, or CER2-like polypeptides, orAt1g68440-like polypeptides and variants of nucleic acids encodingDEAD-box RNA helicase polypeptides, or CER2-like polypeptides, orAt1g68440-like polypeptides obtained by gene shuffling. The termshybridising sequence, splice variant, allelic variant and gene shufflingare as described herein.

Nucleic acids encoding DEAD-box RNA helicase polypeptides, or CER2-likepolypeptides, or At1g68440-like polypeptides need not be full-lengthnucleic acids, since performance of the methods of the invention doesnot rely on the use of full-length nucleic acid sequences. According tothe present invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a portion of any one of the nucleic acid sequences given inTable A1 to A3 of the Examples section, or a portion of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in Table A1 to A3 of the Examples section.

A portion of a nucleic acid may be prepared, for example, by making oneor more deletions to the nucleic acid. The portions may be used inisolated form or they may be fused to other coding (or non-coding)sequences in order to, for example, produce a protein that combinesseveral activities. When fused to other coding sequences, the resultantpolypeptide produced upon translation may be bigger than that predictedfor the protein portion.

Concerning DEAD-box RNA helicase polypeptides, portions useful in themethods of the invention, encode a DEAD-box RNA helicase polypeptide asdefined herein, and have substantially the same biological activity asthe amino acid sequences given in Table A1 of the Examples section.Preferably, the portion is a portion of any one of the nucleic acidsgiven in Table A1 of the Examples section, or is a portion of a nucleicacid encoding an orthologue or paralogue of any one of the amino acidsequences given in Table A1 of the Examples section. Preferably theportion is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250 consecutive nucleotides inlength, the consecutive nucleotides being of any one of the nucleic acidsequences given in Table A1 of the Examples section, or of a nucleicacid encoding an orthologue or paralogue of any one of the amino acidsequences given in Table A1 of the Examples section. Most preferably theportion is a portion of the nucleic acid of SEQ ID NO: 1. Preferably,the portion encodes a fragment of an amino acid sequence which, whenused in the construction of a phylogenetic tree, such as the onedepicted in FIG. 3, clusters with the group of DEAD-box RNA helicasepolypeptides (more preferably, subfamily VI as described by Aubourg etal. (1999)) comprising the amino acid sequence represented by SEQ ID NO:2 rather than with any other group and/or comprises any one of themotifs 1 to 9 as described above and/or has helicase activity and/or hasat least 45% sequence identity to SEQ ID NO: 2.

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a DEAD-box RNA helicase polypeptide as defined herein, or witha portion as defined herein.

Concerning CER2-like polypeptides, portions useful in the methods of theinvention, encode a CER2-like polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A2 of the Examples section. Preferably, the portion is aportion of any one of the nucleic acids given in Table A2 of theExamples section, or is a portion of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A2 of the Examples section. Preferably the portion is at least500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000,consecutive nucleotides in length, the consecutive nucleotides being ofany one of the nucleic acid sequences given in Table A2 of the Examplessection, or of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A2 of the Examplessection. Most preferably the portion is a portion of the nucleic acid ofSEQ ID NO: 168. Preferably, the portion encodes a fragment of an aminoacid sequence which, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 9, clusters with the group ofCER2-like polypeptides of Clade A comprising the amino acid sequencerepresented by SEQ ID NO: 169 rather than with any other group and/orcomprises at least, 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, or all 9 motifs 18a, 19a, 20a, 21a, 22a, 23a,24, 25, or 26 and/or has biological activity of acyltransferases.

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a CER2-like polypeptide as defined herein, or with a portion asdefined herein.

Concerning At1g68440-like polypeptides, portions useful in the methodsof the invention, encode an At1g68440-like polypeptide as definedherein, and have substantially the same biological activity as the aminoacid sequences given in Table A3 of the Examples section. Preferably,the portion is a portion of any one of the nucleic acids given in TableA3 of the Examples section, or is a portion of a nucleic acid encodingan orthologue or paralogue of any one of the amino acid sequences givenin Table A3 of the Examples section. Preferably the portion is at least500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100consecutive nucleotides in length, the consecutive nucleotides being ofany one of the nucleic acid sequences given in Table A3 of the Examplessection, or of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A3 of the Examplessection. Most preferably the portion is a portion of the nucleic acid ofSEQ ID NO: 291. Preferably, the portion encodes a fragment of an aminoacid sequence which, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 14, clusters with the group ofAt1g68440-like polypeptides of Clade A comprising the amino acidsequence represented by SEQ ID NO: 292 rather than with any other groupand/or comprises at least, 2, at least 3, at least 4, at least 5, or all6 motifs selected from 27a, 28a, 29a, 30a, 34a, 35a.

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding an At1g68440-like polypeptide as defined herein, or with aportion as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising reducing orsubstantially eliminating expression in a plant a nucleic acid capableof hybridizing to any one of the nucleic acids given in Table A1 to A3of the Examples section, or comprising introducing and expressing in aplant a nucleic acid capable of hybridising to a nucleic acid encodingan orthologue, paralogue or homologue of any of the nucleic acidsequences given in Table A1 to A3 of the Examples section.

Concerning DEAD-box RNA helicase polypeptides, hybridising sequencesuseful in the methods of the invention encode a DEAD-box RNA helicasepolypeptide as defined herein, having substantially the same biologicalactivity as the amino acid sequences given in Table A1 of the Examplessection. Preferably, the hybridising sequence is capable of hybridisingto the complement of any one of the nucleic acids given in Table A1 ofthe Examples section, or to a portion of any of these sequences, aportion being as defined above, or the hybridising sequence is capableof hybridising to the complement of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A1 of the Examples section. Most preferably, the hybridisingsequence is capable of hybridising to the complement of a nucleic acidas represented by SEQ ID NO: 1 or to a portion thereof.

Preferably, the hybridising sequence encodes a polypeptide with an aminoacid sequence which, when full-length and used in the construction of aphylogenetic tree, such as the one depicted in FIG. 3, clusters with thegroup of DEAD-box RNA helicase polypeptides (more preferably, subfamilyVI as described by Aubourg et al. (1999)) comprising the amino acidsequence represented by SEQ ID NO: 2 rather than with any other groupAND/OR comprises any one of the motifs 1 to 9 as described above AND/ORhas helicase activity AND/OR has at least 45% sequence identity to SEQID NO: 2.

Concerning CER2-like polypeptides, hybridising sequences useful in themethods of the invention encode a CER2-like polypeptide as definedherein, having substantially the same biological activity as the aminoacid sequences given in Table A2 of the Examples section. Preferably,the hybridising sequence is capable of hybridising to the complement ofany one of the nucleic acids given in Table A2 of the Examples section,or to a portion of any of these sequences, a portion being as definedabove, or the hybridising sequence is capable of hybridising to thecomplement of a nucleic acid encoding an orthologue or paralogue of anyone of the amino acid sequences given in Table A2 of the Examplessection. Most preferably, the hybridising sequence is capable ofhybridising to the complement of a nucleic acid as represented by SEQ IDNO: 168 or to a portion thereof.

Preferably, the hybridising sequence encodes a polypeptide with an aminoacid sequence which, when used in the construction of a phylogenetictree, such as the one depicted in FIG. 9, clusters with the group ofCER2-like polypeptides of Clade A comprising the amino acid sequencerepresented by SEQ ID NO: 169 rather than with any other group and/orcomprises at least, 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, or all 9 motifs 18a, 19a, 20a, 21a, 22a, 23a,24, 25, or 26 and/or has biological activity of acyltransferases.

Concerning At1g68440-like polypeptides, hybridising sequences useful inthe methods of the invention encode an At1g68440-like polypeptide asdefined herein, having substantially the same biological activity as theamino acid sequences given in Table A3 of the Examples section.Preferably, the hybridising sequence is capable of hybridising to thecomplement of any one of the nucleic acids given in Table A3 of theExamples section, or to a portion of any of these sequences, a portionbeing as defined above, or the hybridising sequence is capable ofhybridising to the complement of a nucleic acid encoding an orthologueor paralogue of any one of the amino acid sequences given in Table A3 ofthe Examples section. Most preferably, the hybridising sequence iscapable of hybridising to the complement of a nucleic acid asrepresented by SEQ ID NO: 291 or to a portion thereof.

Preferably, the hybridising sequence encodes a polypeptide with an aminoacid sequence which, when full-length and used in the construction of aphylogenetic tree, such as the one depicted in FIG. 14, clusters withthe group of At1g68440-like polypeptides of Clade A comprising the aminoacid sequence represented by SEQ ID NO: 292 rather than with any othergroup and/or comprises at least, 2, at least 3, at least 4, at least 5,or all 6 motifs selected from 27a, 28a, 29a, 30a, 31a, 32a.

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding a DEAD-box RNA helicase polypeptide, or aCER2-like (CER2-like acyl transferase) polypeptide, or an At1g68440-likepolypeptide as defined hereinabove, a splice variant being as definedherein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising reducing orsubstantially eliminating expression in a plant of a splice variant ofany one of the nucleic acid sequences given in Table A1 to A3 of theExamples section, or a splice variant of a nucleic acid encoding anorthologue, paralogue or homologue of any of the amino acid sequencesgiven in Table A1 to A3 of the Examples section.

Concerning DEAD-box RNA helicase polypeptides, preferred splice variantsare splice variants of a nucleic acid represented by SEQ ID NO: 1, or asplice variant of a nucleic acid encoding an orthologue or paralogue ofSEQ ID NO: 2. Preferably, the amino acid sequence encoded by the splicevariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 3, clusters with the group of DEAD-box RNAhelicase polypeptides (more preferably, subfamily VI as described byAubourg et al. (1999)) comprising the amino acid sequence represented bySEQ ID NO: 2 rather than with any other group and/or comprises any oneof the motifs 1 to 9 as described above and/or has helicase activityand/or has at least 45% sequence identity to SEQ ID NO: 2.

Concerning CER2-like polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 168, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 169. Preferably, the amino acid sequence encoded by the splicevariant which, when used in the construction of a phylogenetic tree,such as the one depicted in FIG. 9, clusters with the group of CER2-likepolypeptides of Clade A comprising the amino acid sequence representedby SEQ ID NO: 169 rather than with any other group and/or comprises atleast, 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, or all 9 motifs 18a, 19a, 20a, 21a, 22a, 23a, 24, 25, or 26and/or has biological activity of acyltransferases.

Concerning At1g68440-like polypeptides, preferred splice variants aresplice variants of a nucleic acid represented by SEQ ID NO: 291, or asplice variant of a nucleic acid encoding an orthologue or paralogue ofSEQ ID NO: 292. Preferably, the amino acid sequence encoded by thesplice variant, when used in the construction of a phylogenetic tree,such as the one depicted in FIG. 14, clusters with the group ofAt1g68440-like polypeptides of Clade A comprising the amino acidsequence represented by SEQ ID NO: 292 rather than with any other groupand/or comprises at least, 2, at least 3, at least 4, at least 5, or all6 motifs selected from 27a, 28a, 29a, 30a, 31a, 32a.

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding a DEAD-boxRNA helicase polypeptide, or a CER2-like (CER2-like acyl transferase)polypeptide, or an At1g68440-like polypeptide as defined hereinabove, anallelic variant being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising reducing orsubstantially eliminating expression in a plant of an allelic variant ofany one of the nucleic acids given in Table A1 to A3 of the Examplessection, or comprising reducing or substantially eliminating expressionin a plant an allelic variant of a nucleic acid encoding an orthologue,paralogue or homologue of any of the amino acid sequences given in TableA1 to A3 of the Examples section.

Concerning DEAD-box RNA helicase polypeptides, the polypeptides encodedby allelic variants useful in the methods of the present invention havesubstantially the same biological activity as the DEAD-box RNA helicasepolypeptide of SEQ ID NO: 2 and any of the amino acids depicted in TableA1 of the Examples section. Allelic variants exist in nature, andencompassed within the methods of the present invention is the use ofthese natural alleles. Preferably, the allelic variant is an allelicvariant of SEQ ID NO: 1 or an allelic variant of a nucleic acid encodingan orthologue or paralogue of SEQ ID NO: 2. Preferably, the amino acidsequence encoded by the allelic variant, when used in the constructionof a phylogenetic tree, such as the one depicted in FIG. 3, clusterswith the group of DEAD-box RNA helicase polypeptides (more preferably,subfamily VI as described by Aubourg et al. (1999)) comprising the aminoacid sequence represented by SEQ ID NO: 2 rather than with any othergroup and/or comprises any one of the motifs 1 to 9 as described aboveand/or has helicase activity and/or has at least 45% sequence identityto SEQ ID NO: 2.

Concerning CER2-like polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the CER2-like polypeptideof SEQ ID NO: 169 and any of the amino acids depicted in Table A2 of theExamples section. Allelic variants exist in nature, and encompassedwithin the methods of the present invention is the use of these naturalalleles. Preferably, the allelic variant is an allelic variant of SEQ IDNO: 168 or an allelic variant of a nucleic acid encoding an orthologueor paralogue of SEQ ID NO: 169. Preferably, the amino acid sequenceencoded by the allelic variant which, when used in the construction of aphylogenetic tree, such as the one depicted in FIG. 9, clusters with thegroup of CER2-like polypeptides of Clade A comprising the amino acidsequence represented by SEQ ID NO: 169 rather than with any other groupand/or comprises at least, 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, or all 9 motifs 18a, 19a, 20a, 21a,22a, 23a, 24, 25, or 26 and/or has biological activity ofacyltransferases.

Concerning At1g68440-like polypeptides, the polypeptides encoded byallelic variants useful in the methods of the present invention havesubstantially the same biological activity as the At1g68440-likepolypeptide of SEQ ID NO: 292 and any of the amino acids depicted inTable A3 of the Examples section. Allelic variants exist in nature, andencompassed within the methods of the present invention is the use ofthese natural alleles. Preferably, the allelic variant is an allelicvariant of SEQ ID NO: 291 or an allelic variant of a nucleic acidencoding an orthologue or paralogue of SEQ ID NO: 292. Preferably, theamino acid sequence encoded by the allelic variant, when used in theconstruction of a phylogenetic tree, such as the one depicted in FIG.14, clusters with the group of At1g68440-like polypeptides of Clade Acomprising the amino acid sequence represented by SEQ ID NO: 292 ratherthan with any other group and/or comprises at least, 2, at least 3, atleast 4, at least 5, or all 6 motifs selected from 27a, 28a, 29a, 30a,31a, 32a.

Gene shuffling or directed evolution may also be used to generatevariants of nucleic acids encoding DEAD-box RNA helicase polypeptides,or CER2-like polypeptides, or At1g68440-like polypeptides as definedabove; the term “gene shuffling” being as defined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a variant of any one of the nucleic acid sequencesgiven in Table A1 to A3 of the Examples section, or comprisingintroducing and expressing in a plant a variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in Table A1 to A3 of the Examples section, which variantnucleic acid is obtained by gene shuffling.

Concerning DEAD-box RNA helicase polypeptides, preferably, the aminoacid sequence encoded by the variant nucleic acid obtained by geneshuffling, when used in the construction of a phylogenetic tree such asthe one depicted in FIG. 3, clusters with the group of DEAD-box RNAhelicase polypeptides (more preferably, subfamily VI as described byAubourg et al. (1999)) comprising the amino acid sequence represented bySEQ ID NO: 2 rather than with any other group and/or comprises any oneof the motifs 1 to 9 as described above and/or has helicase activityand/or has at least 45% sequence identity to SEQ ID NO: 2.

Concerning CER2-like polypeptides, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling, whenused in the construction of a phylogenetic tree, such as the onedepicted in FIG. 9, clusters with the group of CER2-like polypeptides ofClade A comprising the amino acid sequence represented by SEQ ID NO: 169rather than with any other group and/or comprises at least, 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9motifs 18a, 19a, 20a, 21a, 22a, 23a, 24, 25, or 26 and/or has biologicalactivity of acyltransferases.

Concerning At1g68440-like polypeptides, preferably, the amino acidsequence encoded by the variant nucleic acid obtained by gene shuffling,when used in the construction of a phylogenetic tree such as the onedepicted in FIG. 14, clusters with the group of At1g68440-likepolypeptides of Clade A comprising the amino acid sequence representedby SEQ ID NO: 292 rather than with any other group and/or comprises atleast, 2, at least 3, at least 4, at least 5, or all 6 motifs selectedfrom 27a, 28a, 29a, 30a, 31a, 32a.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (Current Protocolsin Molecular Biology. Wiley Eds.).

Nucleic acids encoding DEAD-box RNA helicase polypeptides may be derivedfrom any natural or artificial source. The nucleic acid may be modifiedfrom its native form in composition and/or genomic environment throughdeliberate human manipulation. Preferably the DEAD-box RNA helicasepolypeptide-encoding nucleic acid is from a plant, further preferablyfrom a monocotyledonous plant, more preferably from the family Poaceae,most preferably the nucleic acid is from Oryza sativa. In an alternativepreferred embodiment the nucleic acid is from Zea mays.

Nucleic acids encoding CER2-like polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the CER2-like polypeptide-encodingnucleic acid is from a plant, further preferably from a dicotyledonousplant, more preferably from the family Fabaceae, most preferably thenucleic acid is from Medicago truncatula.

Nucleic acids encoding At1g68440-like polypeptides may be derived fromany natural or artificial source. The nucleic acid may be modified fromits native form in composition and/or genomic environment throughdeliberate human manipulation. Preferably the At1g68440-likepolypeptide-encoding nucleic acid is from a plant, further preferablyfrom a dicotyledonous plant, more preferably from the family Salicaceae,most preferably the nucleic acid is from Populus trichocarpa.

Performance of the methods of the invention gives plants having enhancedyield-related traits. In particular performance of the methods of theinvention gives plants having increased yield, especially increased seedyield relative to control plants. The terms “yield” and “seed yield” aredescribed in more detail in the “definitions” section herein.

Reference herein to enhanced yield-related traits is taken to mean anincrease in early vigour and/or in biomass (weight) of one or more partsof a plant, which may include aboveground (harvestable) parts and/or(harvestable) parts below ground. In particular, such harvestable partsare seeds, and performance of the methods of the invention results inplants having increased seed yield relative to the seed yield of controlplants.

The present invention provides a method for increasing yield-relatedtraits, especially seed yield of plants, relative to control plants,which method comprises modulating expression in a plant of a nucleicacid encoding a DEAD-box RNA helicase polypeptide, or a CER2-like(CER2-like acyl transferase) polypeptide, or an At1g68440-likepolypeptide as defined herein.

Since the transgenic plants according to the present invention haveincreased seed yield, it is likely that these plants exhibit anincreased growth rate (during at least part of their life cycle),relative to the growth rate of control plants at a corresponding stagein their life cycle.

Concerning DEAD-box RNA helicase polypeptides, according to a preferredfeature of the present invention, performance of the methods of theinvention gives plants having an increased growth rate relative tocontrol plants. Therefore, according to the present invention, there isprovided a method for increasing the growth rate of plants, which methodcomprises reducing or substantially eliminating expression in a plant ofa nucleic acid encoding a DEAD-box RNA helicase polypeptide as definedherein.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,according to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression in a plant of anucleic acid encoding a CER2-like polypeptide as defined herein.

Concerning DEAD-box RNA helicase polypeptides, performance of themethods of the invention gives plants grown under non-stress conditionsor under mild drought conditions increased yield relative to controlplants grown under comparable conditions. Therefore, according to thepresent invention, there is provided a method for increasing yield inplants grown under non-stress conditions or under mild droughtconditions, which method comprises reducing or substantially eliminatingexpression in a plant of a nucleic acid encoding a DEAD-box RNA helicasepolypeptide.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises modulating expression ina plant of a nucleic acid encoding a CER2-like polypeptide.

Concerning DEAD-box RNA helicase polypeptides, performance of themethods of the invention gives plants grown under conditions of nutrientdeficiency, particularly under conditions of nitrogen deficiency,increased yield relative to control plants grown under comparableconditions. Therefore, according to the present invention, there isprovided a method for increasing yield in plants grown under conditionsof nutrient deficiency, which method comprises reducing or substantiallyeliminating expression in a plant of a nucleic acid encoding a DEAD-boxRNA helicase polypeptide.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesmodulating expression in a plant of a nucleic acid encoding a CER2-likepolypeptide.

Concerning DEAD-box RNA helicase polypeptides, performance of themethods of the invention gives plants grown under conditions of saltstress, increased yield relative to control plants grown undercomparable conditions. Therefore, according to the present invention,there is provided a method for increasing yield in plants grown underconditions of salt stress, which method comprises reducing orsubstantially eliminating expression in a plant of a nucleic acidencoding a DEAD-box RNA helicase polypeptide.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,performance of the methods of the invention gives plants grown underconditions of salt stress, increased yield relative to control plantsgrown under comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of salt stress, which method comprises modulatingexpression in a plant of a nucleic acid encoding a CER2-likepolypeptide.

Concerning DEAD-box RNA helicase polypeptides, performance of themethods of the invention gives plants grown under conditions of droughtstress, increased yield relative to control plants grown undercomparable conditions. Therefore, according to the present invention,there is provided a method for increasing yield in plants grown underconditions of drought stress, which method comprises reducing orsubstantially eliminating expression in a plant of a nucleic acidencoding a DEAD-box RNA helicase polypeptide.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,performance of the methods of the invention gives plants grown underconditions of drought stress, increased yield relative to control plantsgrown under comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of drought stress, which method comprisesmodulating expression in a plant of a nucleic acid encoding a CER2-likepolypeptide.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression in plants of nucleic acids encoding aninverted repeat of DEAD-box RNA helicase polypeptides, or CER2-likepolypeptides, or At1g68440-like polypeptides. The gene constructs may beinserted into vectors, which may be commercially available, suitable fortransforming into plants and suitable for expression of the gene ofinterest in the transformed cells. The invention also provides use of agene construct as defined herein in the methods of the invention.

Concerning DEAD-box RNA helicase polypeptides, more specifically, thepresent invention provides a construct comprising:

-   -   (a) an inverted repeat of a DEAD-box RNA helicase gene or        fragment, capable of downregulating an endogeneous target        DEAD-box RNA helicase;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Preferably, the nucleic acid encoding a DEAD-box RNA helicasepolypeptide is as defined above. The term “control sequence” and“termination sequence” are as defined herein.

Concerning CER2-like polypeptides, more specifically, the presentinvention provides a construct comprising:

-   -   (a) a nucleic acid encoding a CER2-like polypeptide as defined        above;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Preferably, the nucleic acid encoding a CER2-like polypeptide is asdefined above. The term “control sequence” and “termination sequence”are as defined herein.

Concerning At1g68440-like polypeptides, more specifically, the presentinvention provides a construct comprising:

-   -   (a) a nucleic acid encoding an At1g68440-like polypeptide as        defined above;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Preferably, the nucleic acid encoding an At1g68440-like polypeptide isas defined above. The term “control sequence” and “termination sequence”are as defined herein.

The invention furthermore provides plants transformed with a constructas described above. In particular, the invention provides plantstransformed with a construct as described above, which plants haveincreased yield-related traits as described herein.

Plants are transformed with a vector comprising any of the nucleic acidsdescribed above. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells containing the sequence ofinterest. The sequence of interest is operably linked to one or morecontrol sequences (at least to a promoter).

Advantageously, any type of promoter, whether natural or synthetic, maybe used to drive expression of the nucleic acid sequence, but preferablythe promoter is of plant origin. A constitutive promoter is particularlyuseful in the methods. Preferably the constitutive promoter is aubiquitous constitutive promoter of medium strength. See the“Definitions” section herein for definitions of the various promotertypes.

Concerning DEAD-box RNA helicase genes, it should be clear that theapplicability of the present invention is not restricted to the DEAD-boxRNA helicase polypeptide-encoding nucleic acid represented by SEQ ID NO:1, nor is the applicability of the invention restricted to expression ofa DEAD-box RNA helicase polypeptide-encoding nucleic acid when driven bya constitutive promoter.

The constitutive promoter is preferably a medium strength promoter. Morepreferably it is a plant derived promoter, such as a GOS2 promoter or apromoter of substantially the same strength and having substantially thesame expression pattern (a functionally equivalent promoter), morepreferably the promoter is the promoter GOS2 promoter from rice. Furtherpreferably the constitutive promoter is represented by a nucleic acidsequence substantially similar to SEQ ID NO: 156, most preferably theconstitutive promoter is identical to SEQ ID NO: 156. See the“Definitions” section herein for further examples of constitutivepromoters.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Preferably, the construct comprisesan expression cassette comprising a GOS2 promoter, substantially similarto SEQ ID NO: 156, and the nucleic acid encoding the DEAD-box RNAhelicase polypeptide. Furthermore, one or more sequences encodingselectable markers may be present on the construct introduced into aplant.

Concerning CER2-like genes, it should be clear that the applicability ofthe present invention is not restricted to the CER2-likepolypeptide-encoding nucleic acid represented by SEQ ID NO: 168, nor isthe applicability of the invention restricted to expression of aCER2-like polypeptide-encoding nucleic acid when driven by aconstitutive promoter, or when driven by a root-specific promoter.

The constitutive promoter is preferably a medium strength promoter. Morepreferably it is a plant derived promoter, such as a GOS2 promoter or apromoter of substantially the same strength and having substantially thesame expression pattern (a functionally equivalent promoter), morepreferably the promoter is the promoter GOS2 promoter from rice. Furtherpreferably the constitutive promoter is represented by a nucleic acidsequence substantially similar to SEQ ID NO: 289, most preferably theconstitutive promoter is as represented by SEQ ID NO: 289. See the“Definitions” section herein for further examples of constitutivepromoters.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Preferably, the construct comprisesan expression cassette comprising a GOS2 promoter, substantially similarto SEQ ID NO: 289, and the nucleic acid encoding the CER2-likepolypeptide. More preferably, the expression cassette comprises thesequence represented by SEQ ID NO: 290 (pPRO::CER2-like gene::t-zeinsequence). Furthermore, one or more sequences encoding selectablemarkers may be present on the construct introduced into a plant.

Concerning At1g68440-like genes, it should be clear that theapplicability of the present invention is not restricted to theAt1g68440-like polypeptide-encoding nucleic acid represented by SEQ IDNO: 291, nor is the applicability of the invention restricted toexpression of an At1g68440-like polypeptide-encoding nucleic acid whendriven by a constitutive promoter.

The constitutive promoter is preferably a medium strength promoter. Morepreferably it is a plant derived promoter, such as a GOS2 promoter or apromoter of substantially the same strength and having substantially thesame expression pattern (a functionally equivalent promoter), morepreferably the promoter is the promoter GOS2 promoter from rice. Furtherpreferably the constitutive promoter is represented by a nucleic acidsequence substantially similar to SEQ ID NO: 363, most preferably theconstitutive promoter is as represented by SEQ ID NO: 363. See the“Definitions” section herein for further examples of constitutivepromoters.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Preferably, the construct comprisesan expression cassette comprising a GOS2 promoter, substantially similarto SEQ ID NO: 363, and the nucleic acid encoding the At1g68440-likepolypeptide.

Concerning DEAD-box RNA helicase genes, according to a preferred featureof the invention, the modulated expression is reduced or substantiallyeliminated expression. Methods for reducing or eliminating expression ofnucleic acids or genes, or gene products, are well documented in the artand examples are provided in the definitions section.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides,according to a preferred feature of the invention, the modulatedexpression is increased expression. Methods for increasing expression ofnucleic acids or genes, or gene products, are well documented in the artand examples are provided in the definitions section.

As mentioned above, a preferred method for modulating expression of anucleic acid encoding a DEAD-box RNA helicase polypeptide, or aCER2-like (CER2-like acyl transferase) polypeptide, or an At1g68440-likepolypeptide is by introducing and expressing in a plant a nucleic acidencoding a DEAD-box RNA helicase polypeptide, or a CER2-like (CER2-likeacyl transferase) polypeptide, or an At1g68440-like polypeptide; howeverthe effects of performing the method, i.e. enhancing yield-relatedtraits may also be achieved using other well known techniques, includingbut not limited to T-DNA inactivation, TILLING, homologousrecombination. A description of these techniques is provided in thedefinitions section.

Concerning DEAD-box RNA helicase polypeptides, the invention alsoprovides a method for the production of transgenic plants havingenhanced yield-related traits relative to control plants, comprisingreducing or substantially eliminating expression in a plant of anynucleic acid encoding a DEAD-box RNA helicase polypeptide as definedhereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having enhanced yield-related traits,particularly increased seed yield, which method comprises:

-   -   (i) reducing or substantially eliminating expression in a plant        or plant cell a DEAD-box RNA helicase polypeptide-encoding        nucleic acid; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a DEAD-box RNA helicase polypeptide as defined herein.

Concerning CER2-like polypeptides, and At1g68440-like polypeptides, theinvention also provides a method for the production of transgenic plantshaving enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding a CER2-like polypeptide, or an At1g68440-like polypeptide asdefined hereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having enhanced yield-related traits,particularly increased seed yield, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell a        CER2-like nucleic acid encoding a CER2-like polypeptide, or an        At1g68440-like polypeptide or a genetic construct comprising a        CER2-like polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a CER2-like polypeptide, or an At1g68440-like polypeptide asdefined herein.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation. The term “transformation” is described in more detail inthe “definitions” section herein.

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention encompasses plants orparts thereof, including seeds, obtainable by the methods according tothe present invention. The plants or parts thereof comprise a nucleicacid transgene encoding a DEAD-box RNA helicase polypeptide as definedabove. The present invention extends further to encompass the progeny ofa primary transformed or transfected cell, tissue, organ or whole plantthat has been produced by any of the aforementioned methods, the onlyrequirement being that progeny exhibit the same genotypic and/orphenotypic characteristic(s) as those produced by the parent in themethods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a DEAD-box RNA helicase polypeptide, or a CER2-likepolypeptide, or an At1g68440-like polypeptide as defined hereinabove.Preferred host cells according to the invention are plant cells,bacterial, yeast or fungal cells. Host plants for the nucleic acids orthe vector used in the method according to the invention, the expressioncassette or construct or vector are, in principle, advantageously allplants, which are capable of synthesizing the polypeptides used in theinventive method.

The methods of the invention are advantageously applicable to any plant,in particular to any plant as defined herein. Plants that areparticularly useful in the methods of the invention include all plantswhich belong to the superfamily Viridiplantae, in particularmonocotyledonous and dicotyledonous plants including fodder or foragelegumes, ornamental plants, food crops, trees or shrubs. According to apreferred embodiment of the present invention, the plant is a cropplant. Examples of crop plants include but are not limited to chicory,carrot, cassaya, trefoil, soybean, beet, sugar beet, sunflower, canola,alfalfa, rapeseed, linseed, cotton, tomato, potato and tobacco.According to another embodiment of the present invention, the plant is amonocotyledonous plant. Examples of monocotyledonous plants includesugarcane. According to another embodiment of the present invention, theplant is a cereal. Examples of cereals include rice, maize, wheat,barley, millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn,teff, milo and oats.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes,tubers and bulbs, which harvestable parts comprise a recombinant nucleicacid encoding a DEAD-box RNA helicase polypeptide, or a CER2-likepolypeptide, or an At1g68440-like polypeptide. The invention furthermorerelates to products derived, preferably directly derived, from aharvestable part of such a plant, such as dry pellets or powders, oil,fat and fatty acids, starch or proteins.

The present invention also encompasses use of nucleic acids encodingDEAD-box RNA helicase polypeptides as described herein and use of theseDEAD-box RNA helicase polypeptides in enhancing any of theaforementioned yield-related traits in plants. For example, nucleicacids encoding DEAD-box RNA helicase polypeptide, or CER2-likepolypeptide, or At1g68440-like polypeptide described herein, or theDEAD-box RNA helicase polypeptides, or the CER2-like polypeptides, orthe At1g68440-like polypeptides themselves, may find use in breedingprogrammes in which a DNA marker is identified which may be geneticallylinked to polypeptide gene encoding a DEAD-box RNA helicase polypeptide,or a CER2-like polypeptide, or an At1g68440-like polypeptide. Thenucleic acids/genes, or the DEAD-box RNA helicase polypeptides, or theCER2-like polypeptides, or the At1g68440-like polypeptides themselvesmay be used to define a molecular marker. This DNA or protein marker maythen be used in breeding programmes to select plants having enhancedyield-related traits as defined hereinabove in the methods of theinvention. Furthermore, allelic variants of nucleic acid/gene encoding aDEAD-box RNA helicase polypeptide, or a CER2-like polypeptide, or anAt1g68440-like polypeptide may find use in marker-assisted breedingprogrammes. Nucleic acids encoding DEAD-box RNA helicase polypeptides,or CER2-like polypeptides, or At1g68440-like polypeptides may also beused as probes for genetically and physically mapping the genes thatthey are a part of, and as markers for traits linked to those genes.Such information may be useful in plant breeding in order to developlines with desired phenotypes.

Items

-   1. A method for enhancing yield-related traits in plants relative to    control plants, comprising modulating expression in a plant of a    nucleic acid encoding a CER2-like polypeptide, wherein said    CER2-like polypeptide comprises a PF02458 domain and/or possesses    transferase activity.-   2. Method according to item 1, wherein said modulated expression is    effected by introducing and expressing in a plant said nucleic acid    encoding said CER2-like polypeptide.-   3. Method according to item 1 or 2, wherein said enhanced    yield-related traits comprise increased yield and/or early vigour    relative to control plants, and preferably comprise increased    biomass and/or increased seed yield relative to control plants.-   4. Method according to any one of items 1 to 3, wherein said    enhanced yield-related traits are obtained under non-stress    conditions.-   5. Method according to any one of items 1 to 3, wherein said    enhanced yield-related traits are obtained under conditions of    drought stress, salt stress or nitrogen deficiency.-   6. Method according to any of items 1 to 5, wherein said CER2-like    polypeptide comprises one or more of the following motifs:

Motif 18a: (SEQ ID NO: 274)FKCGGL[SA][LI]G[LV][SG]W[AS]HI[LV][GA]D[GA]FSA SHFIN[SA]W, Motif 19a:(SEQ ID NO: 276) SGR[PL]E[IVL]KCNDEG[VA][RL][FI][VI]EAE[CA]D, Motif 20a:(SEQ ID NO: 278) xY[ST][TR][FY]E[AI]L[AST][AG][HL][IV]W[RK][CS]I[AC]KARG

-   7. Method according any of items 1 to 5, wherein said CER2-like    polypeptide comprises

Motif 21a: (SEQ ID NO: 280)VQ[VF]TxFKCGG[LM][SA][LIV]GLS[WC]AH[IVL]LGD[APV] FSA[ST]TF[FIM][NK]KWMotif 22a: (SEQ ID NO: 282) [EA]SGR[PW][YF][IV]KCND[AC]GVRIVEA[KHR]CDKMotif 23a: (SEQ ID NO: 284)SR[VT][GE][EP][GN]Kx[HY]E[LP][ST]x[LM]DLAMKLHY [LVI]R[GA]VY[FY][FY]

-   8. Method according any of items 1 to 5, wherein said CER2-like    polypeptide comprises

Motif 24: (SEQ ID NO: 286)FKCGG[VIF][SA][LI]G[VL]G[MI]SHx[VLM]ADGxSALHFI [NS][SAT]W Motif 25:(SEQ ID NO: 287) NPNLL[IV][TV]SWT[RT][LF]P[ILF][YH][DE]ADFGWG[KR]PI[FY]MGP Motif 26: (SEQ ID NO: 288)[SA]LS[KE][VT]L[VT][HP][FY]YP[ML]AGR.

-   9. Method according to any one of items 1 to 8, wherein said nucleic    acid encoding a CER2-like is of plant origin, preferably from a    dicotyledonous plant, further preferably from the family Fabaceae,    more preferably from the genus Medicago, most preferably from    Medicago truncatula.-   10. Method according to any one of items 1 to 9, wherein said    nucleic acid encoding a CER2-like encodes any one of the    polypeptides listed in Table A2 or is a portion of such a nucleic    acid, or a nucleic acid capable of hybridising with such a nucleic    acid.-   11. Method according to any one of items 1 to 10, wherein said    nucleic acid sequence encodes an orthologue or paralogue of any of    the polypeptides given in Table A2.-   12. Method according to any one of items 1 to 11, wherein said    nucleic acid encoding said CER2-like polypeptide corresponds to SEQ    ID NO: 291.-   13. Method according to any one of items 1 to 12, wherein said    nucleic acid is operably linked to a constitutive promoter,    preferably to a medium strength constitutive promoter, preferably to    a plant promoter, more preferably to a GOS2 promoter, most    preferably to a GOS2 promoter from rice.-   14. Plant, plant part thereof, including seeds, or plant cell,    obtainable by a method according to any one of items 1 to 13,    wherein said plant, plant part or plant cell comprises a recombinant    nucleic acid encoding a CER2-like polypeptide as defined in any of    items 1 and 5 to 12.-   15. Construct comprising:    -   (i) nucleic acid encoding a CER2-like as defined in any of items        1 and 6 to 12;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (i); and optionally    -   (iii) a transcription termination sequence.-   16. Construct according to item 15, wherein one of said control    sequences is a constitutive promoter, preferably a medium strength    constitutive promoter, preferably to a plant promoter, more    preferably a GOS2 promoter, most preferably a GOS2 promoter from    rice.-   17. Use of a construct according to item 15 or 16 in a method for    making plants having enhanced yield-related traits, preferably    increased yield relative to control plants, and more preferably    increased seed yield and/or increased biomass relative to control    plants.-   18. Plant, plant part or plant cell transformed with a construct    according to item 15 or 16.-   19. Method for the production of a transgenic plant having enhanced    yield-related traits relative to control plants, preferably    increased yield relative to control plants, and more preferably    increased seed yield and/or increased biomass relative to control    plants, comprising:    -   (i) introducing and expressing in a plant cell or plant a        nucleic acid encoding a CER2-like polypeptide as defined in any        of items 1 and 5 to 12; and    -   (ii) cultivating said plant cell or plant under conditions        promoting plant growth and development.-   20. Transgenic plant having enhanced yield-related traits relative    to control plants, preferably increased yield relative to control    plants, and more preferably increased seed yield and/or increased    biomass, resulting from modulated expression of a nucleic acid    encoding a CER2-like polypeptide as defined in any of items 1 and 6    to 12 or a transgenic plant cell derived from said transgenic plant.-   21. Transgenic plant according to item 14, 18 or 20, or a transgenic    plant cell derived therefrom, wherein said plant is a crop plant,    such as beet, sugarbeet or alfalfa; or a monocotyledonous plant such    as sugarcane; or a cereal, such as rice, maize, wheat, barley,    millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn,    teff, milo or oats.-   22. Harvestable parts of a plant according to item 21, wherein said    harvestable parts are preferably shoot biomass and/or seeds.-   23. Products derived from a plant according to item 21 and/or from    harvestable parts of a plant according to item 22.-   24. Use of a nucleic acid encoding a CER2-like polypeptide as    defined in any of items 1 and 6 to 12 for enhancing yield-related    traits in plants relative to control plants, preferably for    increasing yield, and more preferably for increasing seed yield    and/or for increasing biomass in plants relative to control plants.-   25. A method for enhancing yield-related traits in plants relative    to control plants, comprising modulating expression in a plant of a    nucleic acid encoding an At1g68440-like polypeptide, wherein said    At1g68440-like polypeptide comprises a transmembrane domain.-   26. Method according to item 25, wherein said modulated expression    is effected by introducing and expressing in a plant said nucleic    acid encoding said At1g68440-like polypeptide.-   27. Method according to item 25 or 26, wherein said enhanced    yield-related traits comprise increased yield relative to control    plants, and preferably comprise increased biomass and/or increased    seed yield relative to control plants.-   28. Method according to any one of items 25 to 27, wherein said    enhanced yield-related traits are obtained under non-stress    conditions.-   29. Method according to any one of items 25 to 27, wherein said    enhanced yield-related traits are obtained under conditions of    drought stress, salt stress or nitrogen deficiency.-   30. Method according to any of items 25 to 29, wherein said    At1g68440-like polypeptide comprises one or more of the following    motifs:

Motif 26a: (SEQ ID NO: 364) Y[SN]F[WC][KT]WGALILA[LV][FVL]A, Motif 27a:(SEQ ID NO: 366) ME[IV][PT][VE][IL]N[RL]I[SG]DF, Motif 28a:(SEQ ID NO: 368) [SN]VV[KQ]LWD[SN]LG[LF]

-   31. Method according to any one of items 25 to 30, wherein said    nucleic acid encoding an At1g68440-like is of plant origin,    preferably from a dicotyledonous plant, further preferably from the    family Salicaceae, more preferably from the genus Populus, most    preferably from Populus trichocarpa.-   32. Method according to any one of items 25 to 30, wherein said    nucleic acid encoding an At1g68440-like encodes any one of the    polypeptides listed in Table A3 or is a portion of such a nucleic    acid, or a nucleic acid capable of hybridising with such a nucleic    acid.-   33. Method according to any one of items 25 to 32, wherein said    nucleic acid sequence encodes an orthologue or paralogue of any of    the polypeptides given in Table A3.-   34. Method according to any one of items 25 to 33, wherein said    nucleic acid encoding said At1g68440-like polypeptide corresponds to    SEQ ID NO: 291.-   35. Method according to any one of items 25 to 34, wherein said    nucleic acid is operably linked to a constitutive promoter,    preferably to a medium strength constitutive promoter, preferably to    a plant promoter, more preferably to a GOS2 promoter, most    preferably to a GOS2 promoter from rice.-   36. Plant, plant part thereof, including seeds, or plant cell,    obtainable by a method according to any one of items 25 to 34,    wherein said plant, plant part or plant cell comprises a recombinant    nucleic acid encoding an At1g68440-like polypeptide as defined in    any of items 25 and 30 to 34.-   37. An isolated nucleic acid molecule selected from:    -   a nucleic acid represented by SEQ ID NO: 291;    -   the complement of a nucleic acid represented by SEQ ID NO: 291;    -   a nucleic acid encoding an At1g68440-like polypeptide having in        increasing order of preference at least 50%, 51%, 52%, 53%, 54%,        55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,        68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,        81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino        acid sequence represented by SEQ ID NO: 292, and additionally or        alternatively comprising one or more motifs having in increasing        order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to        any one or more of the motifs given in SEQ ID NO: 364 to SEQ ID        NO: 375, and further preferably conferring enhanced        yield-related traits relative to control plants.    -   a nucleic acid molecule which hybridizes with a nucleic acid        molecule of (i) to (iii) under high stringency hybridization        conditions and preferably confers enhanced yield-related traits        relative to control plants.    -   a nucleic acid encoding an At1g68440-like polypeptide having, in        increasing order of preference, at least 50%, 51%, 52%, 53%,        54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,        67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the        amino acid sequence represented by SEQ ID NO: 292 and any of the        other amino acid sequences in Table A3 and preferably conferring        enhanced yield-related traits relative to control plants.-   38. An isolated polypeptide selected from:    -   an amino acid sequence represented by SEQ ID NO: 292;    -   an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by SEQ ID NO: 2, and additionally or alternatively        comprising one or more motifs having in increasing order of        preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,        95%, 96%, 97%, 98%, 99% or more sequence identity to any one or        more of the motifs given in SEQ ID NO: 364 to SEQ ID NO: 375,        and/or any of the other amino acid sequences in Table A3, and        further preferably conferring enhanced yield-related traits        relative to control plants;    -   derivatives of any of the amino acid sequences given in (i)        or (ii) above.-   39. Construct comprising:    -   nucleic acid encoding an At1g68440-like as defined in any of        items 25 and 30 to 34;    -   one or more control sequences capable of driving expression of        the nucleic acid sequence of (i); and optionally    -   a transcription termination sequence.-   40. Construct according to item 39, wherein one of said control    sequences is a constitutive promoter, preferably a medium strength    constitutive promoter, preferably to a plant promoter, more    preferably a GOS2 promoter, most preferably a GOS2 promoter from    rice.-   41. Use of a construct according to item 39 or 40 in a method for    making plants having enhanced yield-related traits, preferably    increased yield relative to control plants, and more preferably    increased seed yield and/or increased biomass relative to control    plants.-   42. Plant, plant part or plant cell transformed with a construct    according to item 39 or 40.-   43. Method for the production of a transgenic plant having enhanced    yield-related traits relative to control plants, preferably    increased yield relative to control plants, and more preferably    increased seed yield and/or increased biomass relative to control    plants, comprising:    -   introducing and expressing in a plant cell or plant a nucleic        acid encoding an At1g68440-like polypeptide as defined in any of        items 25 and 30 to 34; and    -   cultivating said plant cell or plant under conditions promoting        plant growth and development.-   44. Transgenic plant having enhanced yield-related traits relative    to control plants, preferably increased yield relative to control    plants, and more preferably increased seed yield and/or increased    biomass, resulting from modulated expression of a nucleic acid    encoding an At1g68440-like polypeptide as defined in any of items 25    and 30 to 34 or a transgenic plant cell derived from said transgenic    plant.-   45. Transgenic plant according to item 36, 42 or 44, or a transgenic    plant cell derived therefrom, wherein said plant is a crop plant,    such as beet, sugarbeet or alfalfa; or a monocotyledonous plant such    as sugarcane; or a cereal, such as rice, maize, wheat, barley,    millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn,    teff, milo or oats.-   46. Harvestable parts of a plant according to item 45, wherein said    harvestable parts are preferably shoot biomass and/or seeds.-   47. Products derived from a plant according to item 45 and/or from    harvestable parts of a plant according to item 46.-   48. Use of a nucleic acid encoding an At1g68440-like polypeptide as    defined in any of items 25 and 30 to 34 for enhancing yield-related    traits in plants relative to control plants, preferably for    increasing yield, and more preferably for increasing seed yield    and/or for increasing biomass in plants relative to control plants.-   49. A method for enhancing yield-related traits in plants relative    to control plants, comprising reducing or substantially eliminating    expression in a plant of a nucleic acid encoding a DEAD-box RNA    helicase polypeptide and/or the level and/or the activity of a    DEAD-box RNA helicase polypeptide in said plant, wherein said    DEAD-box RNA helicase polypeptide comprises signature pattern    [LIVMF]-[LIVMFFD-E-A-D4RKEN]-X-[LIVMFYGSTN] (SEQ ID NO: 159).-   50. Method according to item 49, wherein said DEAD-box RNA helicase    polypeptide is an orthologue or paralogue to subfamily VI of the 6    subfamilies of DEAD-box RNA helicases as described by Aubourg et al.    (1999).-   51. Method according to item 49 or 50, wherein said DEAD-box RNA    helicase polypeptide comprises one or more of the following motifs:

(i) Motif 1: (SEQ ID NO: 3) [LM][VI]ATDVA[AS]RGLD[IV][KP]D[VI][EK]VV[IV]N[YF][SD][YF]P[LN][TD][IT][ED]DYVHRIGRTGRA, (ii) Motif 2: (SEQ ID NO: 4)[RSN]L[NR][DR]V[TS][YF][LV]VLDEADRMLDMGFEP [EQ][IV]R[AK]I[VL], (iii)Motif 3: (SEQ ID NO: 5) P[TS]PIQA[YQ][AS][WI]P[YI][AL][LM][DS]GRD[FL][IV][GA]IA[KA]TGSGKT

-   52. Method according to any of the items 49 to 51, wherein said    nucleic acid encoding a DEAD-box RNA helicase polypeptide encodes    any one of the proteins listed in Table A1 or is a portion of such a    nucleic acid, or a nucleic acid capable of hybridising with such a    nucleic acid.-   53. Method according to any one of items 49 to 52, wherein said    nucleic acid sequence encodes an orthologue or paralogue of any of    the proteins given in Table A1.-   54. Method according to any of the items 49 to 53, wherein said    reduction or substantial elimination is effected by RNA-mediated    downregulation of gene expression.-   55. Method according to item 54, wherein said RNA-mediated    downregulation is effected by co-suppression.-   56. Method according to item 55, wherein said RNA-mediated    downregulation is effected by use of antisense DEAD-box RNA helicase    nucleic acid sequences.-   57. Method according to any of the items 49 to 53, wherein said    reduction or substantial elimination is effected by using an    inverted repeat of a DEAD-box RNA helicase nucleic acid sequences or    fragment thereof.-   58. Method according to any of the items 49 to 53, wherein said    reduction or substantial elimination is effected using a microRNA.-   59. Method according to any of the items 49 to 53, wherein said    reduction or substantial elimination is effected by insertion    mutagenesis.-   60. Method according to any of the items 49 to 59, comprising    introduction into a host plant a DEAD-box RNA helicase nucleic acid    or a fragment thereof substantially homologous to said nucleic acid    encoding a DEAD-box RNA helicase polypeptide.-   61. Method according to any of the items 49 to 59, wherein said    reduction or substantial elimination is effected in a constitutive    manner preferably by using a constitutive promoter, more preferably    a GOS2 promoter, most preferably to a GOS2 promoter from rice.-   62. Method according to item 60 or 61, wherein said introduced    nucleic acid is from the same family, more preferably from the same    genus, even more preferably from same species as the host plant.-   63. Method according to any of the items 60 to 62, wherein said    introduced DEAD-box RNA helicase nucleic acid sequence comprises a    sufficient length of substantially contiguous nucleotides of SEQ ID    NO: 1 or an orthologue or paralogue thereof and wherein said host    plant is a cereal, preferably rice.-   64. Method according to item 63, wherein said nucleic acid encodes a    protein as represented by any one of the SEQ ID NO: 13, 15, 17, 19,    21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,    55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,    89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,    119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,    145, 147, 149, 151, 153, 155.-   65. Method according to item 63 or 64, wherein said nucleic acid is    a represented by any of SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26,    28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,    62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,    96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,    124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,    150, 152, 154.-   66. Method according to any of items 49 to 65, wherein said enhanced    yield-related traits is increased seed yield relative to control    plants.-   67. Method according to item 66, wherein said increased seed yield    is selected from one or more of the following: a) increased seed    biomass, i.e. seed weight; b) increased number of filled seeds; c)    increased fill rate and d) increased harvest index.-   68. Method according to any of the items 49 to 67, wherein said    increased yield is obtained under non-stress conditions.-   69. Method according to any or the items 49 to 67, wherein said    increased yield is obtained under conditions of drought stress, salt    stress or nitrogen deficiency.-   70. Plant or part thereof, including seeds, obtainable by a method    according to any one of the items 49 to 69, wherein said plant or    part thereof comprises a recombinant nucleic acid encoding a    DEAD-box RNA helicase polypeptide.-   71. Construct comprising:    -   (a) an inverted repeat of a DEAD-box RNA helicase gene or        fragment, capable of downregulating an endogeneous target        DEAD-box RNA helicase;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) transcription termination sequence.-   72. Construct according to item 71, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.-   73. Use of a construct according to item 71 or 72 in a method for    making plants having increased yield, particularly increased biomass    and/or increased seed yield relative to control plants.-   74. Plant, plant part or plant cell transformed with a construct    according to item 71 or 72.-   75. Method for the production of a transgenic plant having increased    yield, particularly increased seed yield relative to control plants,    comprising:    -   (i) introducing and expressing in a plant cell the construct        according to item 71 or 72; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.-   76. Use of DEAD-box RNA helicase nucleic acids for the reduction or    substantial elimination of endogenous DEAD-box gene expression in    plant to increase yield in plants relative to control plants.-   77. Use according to item 76, wherein said increased yield is    increased seed yield.-   78. Use according to item 77, wherein said increased seed yield is    selected from one or more of the following: a) increased seed    biomass, i.e. seed weight; b) increased number of filled seeds; c)    increased fill rate and d) increased harvest index.-   79. Use according to any one of items 76 to 78, wherein said yield    increase occurs under stress conditions.-   80. An isolated nucleic acid molecule selected from:    -   (a) a nucleic acid represented by SEQ ID NO: 12 or 14;    -   (b) the complement of a nucleic acid represented by SEQ ID NO:        12 or 14;    -   (c) a nucleic acid encoding a DEAD-box RNA helicase polypeptide        having in increasing order of preference at least 50%, 51%, 52%,        53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,        66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,        79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,        92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to        the amino acid sequence represented by SEQ ID NO: 13 or 15, and        additionally or alternatively comprising one or more motifs        having in increasing order of preference at least 50%, 55%, 60%,        65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more        sequence identity to any one or more of the motifs given in SEQ        ID NO: 3 to SEQ ID NO: 11, and further preferably conferring        enhanced yield-related traits relative to control plants.    -   (d) a nucleic acid molecule which hybridizes with a nucleic acid        molecule of (i) to (iii) under high stringency hybridization        conditions and preferably confers enhanced yield-related traits        relative to control plants.-   81. An isolated polypeptide selected from:    -   (i) an amino acid sequence represented by SEQ ID NO: 13 or 15;    -   (ii) an amino acid sequence having, in increasing order of        preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,        58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% sequence identity to the amino acid sequence        represented by SEQ ID NO: 13 or 15, and additionally or        alternatively comprising one or more motifs having in increasing        order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to        any one or more of the motifs given in SEQ ID NO: 3 to SEQ ID        NO: 11, and further preferably conferring enhanced yield-related        traits relative to control plants;    -   (iii) derivatives of any of the amino acid sequences given        in (i) or (ii) above.-   82. Transgenic plant having increased yield, particularly increased    biomass and/or increased seed yield, relative to control plants,    resulting from reduced or substantially eliminated expression of a    nucleic acid encoding a DEAD-box RNA helicase polypeptide as defined    in item 49 or 50, or a transgenic plant cell derived from said    transgenic plant.-   83. Transgenic plant according to item 70 or 74, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant, such    as beet, sugarbeet or alfalfa, or a monocot such as sugarcane, or a    cereal, such as rice, maize, wheat, barley, millet, rye, triticale,    sorghum emmer, spelt, secale, einkorn, teff, milo and oats.-   84. Harvestable parts of a plant according to item 82 and 83,    wherein said harvestable parts are preferably shoot biomass and/or    seeds.-   85. Products derived from a plant according to item 82 and 83 and/or    from harvestable parts of a plant according to item 84.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 represents the domain structure of SEQ ID NO: 2 with conservedMEME motifs

FIG. 2 represents a multiple alignment of various DEAD-box RNA helicasepolypeptides of Glade A as shown in FIG. 3. The asterisks indicateidentical amino acids among the various protein sequences, colonsrepresent highly conserved amino acid substitutions, and the dotsrepresent less conserved amino acid substitution; on other positionsthere is no sequence conservation. These alignments can be used fordefining further motifs, when using conserved amino acids.

FIG. 3 shows phylogenetic tree of DEAD-box RNA helicase polypeptides,protein sequences used for constructing the tree are referenced byGenbank Accession numbers as described in FIG. 4 of Auburg et al.(1999).

FIG. 4 shows the MATGAT table for Glade A as shown in FIG. 3.

FIG. 5 represents the binary vector used for Os DEAD-box RNA helicaseRNA silencing in Oryza sativa, using a hairpin construct under thecontrol of a rice GOS2 promoter (pGOS2).

FIG. 6 shows the neighbour-joining phylogenetic tree DEAD-box RNAhelicase family in Arabidopsis as published by Auburg et al. (1999). Thedifferent subfamilies in which the DEAD-box RNA helicases areclassified, including subfamily VI are indicated.

FIG. 7 represents the domain structure of SEQ ID NO: 169 with conservedmotifs or domains.

FIG. 8 represents a multiple alignment of various CER2-likepolypeptides. These alignments can be used for defining further motifs,when using conserved amino acids.

FIG. 9 shows phylogenetic tree of CER2-like polypeptides, Phylogeneticrelationship of CER-like related proteins. The proteins were alignedusing MAFT (Katoh and Toh (2008). Briefings in Bioinformatics9:286-298). A neighbour-joining tree was calculated using QuickTree1.1(Houwe et al. (2002). Bioinformatics 18(11):1546-7). A cladodrogram wasdrawn using Dendroscope2.0.1 (Huson et al. (2007). Bioinformatics8(1):460). At e=1e-10, Arabidopsis transferase genes were recovered. Thetree was generated using representative members of each cluster. Thepreferred CER2-like polypeptide is M. truncatula_transferase_CER2-like#1gene from Medicago truncatula and its closely related homolog areindicated in red lines and highlighted in yellow in the tree.Arabidopsis CER2 and maize Glossy2 both involved in cuticlar waxbiosynthesis are highlighted in green.

FIG. 10 shows the MATGAT table (Example 3)

FIG. 11 represents the binary vector used for increased expression inOryza sativa of a CER2-like-encoding nucleic acid under the control of arice GOS2 promoter (pGOS2).

FIG. 12 represents the domain structure of SEQ ID NO: 292 with conservedmotifs or domains.

FIG. 13 represents a multiple alignment of various At1g68440-likepolypeptides. The asterisks indicate identical amino acids among thevarious protein sequences, colons represent highly conserved amino acidsubstitutions, and the dots represent less conserved amino acidsubstitution; on other positions there is no sequence conservation.These alignments can be used for defining further motifs, when usingconserved amino acids.

FIG. 14 shows phylogenetic tree of At1g68440-like polypeptides,

FIG. 15 shows the MATGAT table (Example 3)

FIG. 16 represents the binary vector used for increased expression inOryza sativa of a At1g68440-like-encoding nucleic acid under the controlof a rice GOS2 promoter (pGOS2).

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration only. The followingexamples are not intended to limit the scope of the invention.

DNA Manipulation

unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in (Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994),Current Protocols in Molecular Biology, Current Protocols. Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfax (1993) by R. D. D. Croy, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

Example 1 Identification of Sequences Related to the Nucleic AcidSequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 1and SEQ ID NO: 2, or to SEQ ID NO: 168 and SEQ ID NO: 169, or to SEQ IDNO: 291 and SEQ ID NO: 292, were identified amongst those maintained inthe Entrez Nucleotides database at the National Center for BiotechnologyInformation (NCBI) using database sequence search tools, such as theBasic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.215:403-410; and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402). The program is used to find regions of local similaritybetween sequences by comparing nucleic acid or polypeptide sequences tosequence databases and by calculating the statistical significance ofmatches. For example, the polypeptide encoded by the nucleic acid of SEQID NO: 1, or SEQ ID NO: 168, or SEQ ID NO: 291 was used for the TBLASTNalgorithm, with default settings and the filter to ignore low complexitysequences set off. The output of the analysis was viewed by pairwisecomparison, and ranked according to the probability score (E-value),where the score reflect the probability that a particular alignmentoccurs by chance (the lower the E-value, the more significant the hit).In addition to E-values, comparisons were also scored by percentageidentity. Percentage identity refers to the number of identicalnucleotides (or amino acids) between the two compared nucleic acid (orpolypeptide) sequences over a particular length. In some instances, thedefault parameters may be adjusted to modify the stringency of thesearch. For example the E-value may be increased to show less stringentmatches. This way, short nearly exact matches may be identified.

1. DEAD-Box RNA Helicase Polypeptide

Table A1 provides a list of nucleic acid sequences related to SEQ ID NO:1 and SEQ ID NO: 2.

TABLE A1 Examples of DEAD-box RNA helicase nucleic acids andpolypeptides: Nucleic acid Protein Plant Source Name SEQ ID NO: SEQ IDNO: O. sativa 1 2 G. max 12 13 Z. mays 14 15 A. thaliana AT1G31970 16 17Aquilegia_sp TC27171 18 19 Chlorella 36420 20 21 G. max Glyma01g01390 2223 G. raimondii TC4727 24 25 H. vulgare TC183460 26 27 M. truncatulaCU459038_20.4 28 29 O. lucimarinus 38084 30 31 O.RCC809 33138 32 33 O.taurii 19637 34 35 P. patens 205380 36 37 S. bicolor Sb02g009590.1 38 39V. carteri 103394 40 41 V. vinifera GSVIVT00003606001 42 43 A. thalianaAT1G55150 44 45 A. thaliana AT5G63120 46 47 Aquilegia_sp TC22818 48 49C. reinhardtii 136376 50 51 G. hirsutum TC112143 52 53 G. maxGlyma08g20670 54 55 G. max Glyma17g09270 56 57 O.RCC809 31039 58 59 O.sativa Os01g10050 60 61 P. tricornutum 17862 62 63 S. bicolorSb03g043450 64 65 A.thaliana_(—) AT3G01540 66 67 A.thaliana_(—)AT3G06480 68 69 B. napus TC64297 70 71 E. huxleyi 429118 72 73 G. maxGlyma19g00260 74 75 H. vulgare TC193227 76 77 O.RCC809 50761 78 79 O.sativa Os01g07740 80 81 O. sativa Os01g36860 82 83 O. sativa Os11g4624084 85 P. patens 180402 86 87 S. lycopersicum TC207089 88 89 G. maxGlyma19g40510 90 91 A. thaliana AT1G20920 92 93 A. thaliana AT2G47330 9495 C. vulgaris 33424 96 97 C. vulgaris 39851 98 99 G. max Glyma01g43960100 101 O. lucimarinus 34710 102 103 O. sativa Os08g06344 104 105 P.patens 147234 106 107 P. patens 149955 108 109 P. patens 182655 110 111P. trichocarpa 715705 112 113 S. bicolor Sb01g037410 114 115 A. thalianaAT3G18600 116 117 A. thaliana AT3G58510 118 119 A. thaliana AT5G65900120 121 A. thaliana AT2G33730 122 123 C. reinhardtii 136917 124 125 C.vulgaris 82742 126 127 G. max Glyma02g26630 128 129 G. max Glyma17g12460130 131 G. max Glyma18g00370 132 133 O. lucimarinus 87146 134 135 O.sativa Os06g48750 136 137 O. sativa Os11g38670 138 139 P. patens 168022140 141 P. trichocarpa 818968 142 143 S. bicolor Sb10g023440 144 145 S.lycopersicum TC195044 146 147 T. aestivum TC315501 148 149 V. carteri82651 150 151 O. sativa Os02g05330 152 153 P. trichocarpa 775952 154 155

2. CER2-Like Polypeptides

Table A2 provides a list of nucleic acid sequences related to SEQ ID NO:168 and SEQ ID NO: 169.

TABLE A2 Examples of CER2-like nucleic acids and polypeptides: Nucleicacid Protein SEQ SEQ Plant Source ID NO ID NO:M.truncatula_transferase_CER2-like#1 168 169 M.truncatula_CU137640_9.3#1170 171 G.max_GM06MC00033_47170619@33#1 172 173 G.max_Glyma02g37870.1#1174 175 P.trichocarpa_558994#1 176 177 Aquilegia_sp_TC23512#1 178 179B.napus_BN06MC34354_BNP3363_30@34198#1 180 181 A.thaliana_AT4G24510.1#1182 183 A.thaliana_AT3G23840.1#1 184 185 A.thaliana_AT4G13840.1#1 186187 G.max_Glyma08g11560.1#1 188 189 P.trichocarpa_549793#1 190 191O.basilicum_TA1387_39350#1 192 193 O.sativa_LOC_Os04g52164.1#1 194 195T.aestivum_TC294447#1 196 197 A.thaliana_AT4G29250.1#1 198 199G.max_Glyma17g31040.1#1 200 201 P.taeda_TA11048_3352#1 202 203G.max_Glyma03g40670.1#1 204 205 Z.mays_ZM07MC26485_BFb0067H12@26407#1206 207 P.trichocarpa_757078#1 208 209 ZmGlossy2 210 211A.thaliana_AT1G27620.1#1 212 213 A.thaliana_AT2G19070.1#1 214 215A.thaliana_AT5G41040.1#1 216 217 A.thaliana_AT5G48930.1#1 218 219A.thaliana_AT5G63560.1#1 220 221 Aquilegia_sp_TC20397#1 222 223B.napus_BN06MC25915_51267477@25820#1 224 225 B.napus_TC72743#1 226 227G.hirsutum_TC130878#1 228 229 G.max_Glyma05g38290.1#1 230 231G.max_Glyma08g42490.1#1 232 233 G.max_Glyma13g44830.1#1 234 235G.max_Glyma17g06860.1#1 236 237 G.max_GM06MC27564_sae62b11@26939#1 238239 L.sativa_TC19194#1 240 241 M.esculenta_TA8094_3983#1 242 243M.truncatula_AC148816_16.4#1 244 245 P.trichocarpa_579978#1 246 247P.trichocarpa_587193#1 248 249 P.trichocarpa_768990#1 250 251P.trichocarpa_784746#1 252 253 P.trichocarpa_807676#1 254 255P.trichocarpa_810871#1 256 257 P.trifoliata_TA5574_37690#1 258 259P.vulgaris_TC13456#1 260 261 S.lycopersicum_TC201861#1 262 263V.vinifera_GSVIVT00017176001#1 264 265 V.vinifera_GSVIVT00035108001#1266 267 S.moellendorffii_431283#1 268 269 P.patens_TC44371#1 270 2713. At1g68440-Like Polypeptides

Table A3 provides a list of nucleic acid sequences related to SEQ ID NO:291 and SEQ ID NO: 292.

TABLE A3 Examples of At1g68440-like nucleic acids and polypeptides(Examples highlighted in grey refer to the group of Clade A (SEQ ID NOs:291-320)): Nucleic acid Protein SEQ SEQ Plant Source ID NO: ID NO:At1g68440-like (Pt putative IMP dehydrogenase) 291 292C.clementina_DY272345#1 293 294 C.clementina_TC10465#1 295 296C.clementina_TC18948#1 297 298 C.clementina_TC31598#1 299 300G.hirsutum_TC134197#1 301 302 P.trichocarpa_769638#1 303 304P.trichocarpa_832731#1 305 306 P.trifoliata_TA5851_37690#1 307 308R.communis_TA1965_3988#1 309 310 A.lyrata_475964#1 311 312A.lyrata_921679#1 313 314 A.thaliana_AT1G25400.1#1 315 316A.thaliana_AT1G68440.1#1 317 318 Aquilegia_sp_TC24194#1 319 320Aquilegia_sp_TC25879#1 321 322 G.max_Glyma10g41960.1#1 323 324G.max_TC278929#1 325 326 G.max_TC339602#1 327 328H.paradoxus_TA2948_73304#1 329 330 H.petiolaris_DY945986#1 331 332L.japonicus_TC42314#1 333 334 L.saligna_DW067807#1 335 336L.sativa_TC23070#1 337 338 L.serriola_TC6013#1 339 340Lvirosa_DW158350#1 341 342 M.domestica_TC34673#1 343 344M.truncatula_AC135604_14.4#1 345 346 N.tabacum_TC41632#1 347 348O.basilicum_TA1347_39350#1 349 350 P.vulgaris_TC13663#1 351 352S.lycopersicum_TC193481#1 353 354 T.officinale_TA2631_50225#1 355 356T.versicolor_TC2688#1 357 358 V.vinifera_GSVIVT00030351001#1 359 360

Sequences have been tentatively assembled and publicly disclosed byresearch institutions, such as The Institute for Genomic Research (TIGR;beginning with TA). The Eukaryotic Gene Orthologs (EGO) database may beused to identify such related sequences, either by keyword search or byusing the BLAST algorithm with the nucleic acid sequence or polypeptidesequence of interest. Special nucleic acid sequence databases have beencreated for particular organisms, such as by the Joint Genome Institute.Furthermore, access to proprietary databases, has allowed theidentification of novel nucleic acid and polypeptide sequences.

Example 2 Alignment of Sequences Related to the Polypeptide SequencesUsed in the Methods of the Invention 1. DEAD-Box RNA HelicasePolypeptide

Alignment of polypeptide sequences was performed using MAFT (Katoh etal. (2008), Briefings in Bioinformatics 9:286-298).

Alignment of polypeptide sequences was performed using the ClustalW 2.0algorithm of progressive alignment (Thompson et al. (1997), NucleicAcids Res 25:4876-4882; Chema et al. (2003), Nucleic Acids Res31:3497-3500) with standard setting (slow alignment, similarity matrix:Gonnet or Blosum 62 (if polypeptides are aligned), gap opening penalty10, gap extension penalty: 0.2. Minor manual editing was done to furtheroptimise the alignment. The DEAD-box RNA helicase polypeptides arealigned in FIG. 2.

A phylogenetic tree of DEAD-box RNA helicase polypeptides (FIG. 3) wasconstructed using a neighbour-joining clustering algorithm as providedin QuickTree 1.1 (Houwe et al. (2002). Bioinformatics 18(11):1546-7)using Dendroscope2.0.1 (Huson et al. (2007). Bioinformatics 8(1):460).The proteins were aligned using MAFT (Katoh and Toh (2008). Briefings inBioinformatics 9:286-298).

2. CER2-Like Polypeptides

A phylogenetic tree of CER2-like polypeptides (FIG. 9) was constructedusing a neighbour-joining clustering algorithm as provided in the AlignXprogramme from the Vector NTI (Invitrogen).

The alignment was generated using MAFFT (Katoh and Toh (2008)—Briefingsin Bioinformatics 9:286-298). A neighbour-joining tree was calculatedusing Quick-Tree (Howe et al. (2002), Bioinformatics 18(11): 1546-7),100 bootstrap repetitions. The circular phylogram was drawn usingDendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460).Confidence for 100 bootstrap repetitions is indicated for majorbranching.

3. At1g68440-Like Polypeptides

A phylogenetic tree of At1g68440-like polypeptides (FIG. 14) wasconstructed using a neighbour-joining clustering algorithm as providedin the AlignX programme from the Vector NTI (Invitrogen).

The alignment was generated using MAFFT (Katoh and Toh (2008)—Briefingsin Bioinformatics 9:286-298). A neighbour-joining tree was calculatedusing Quick-Tree (Howe et al. (2002), Bioinformatics 18(11): 1546-7),100 bootstrap repetitions. The circular phylogram was drawn usingDendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460)—FIG. 13.Confidence for 100 bootstrap repetitions is indicated for majorbranching.

Example 3 Calculation of Global Percentage Identity Between PolypeptideSequences Useful in Performing the Methods of the Invention

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.

1. DEAD-Box RNA Helicase Polypeptide

As an example, results of the software analysis of selected DEAD-box RNAhelicases of Table A1, i.e. the sequences of Glade A in FIG. 3, areshown in FIG. 4 for the global similarity and identity over the fulllength of the polypeptide sequences. Sequence similarity is shown in thebottom half of the dividing line and sequence identity is shown in thetop half of the diagonal dividing line. Parameters used in thecomparison were: Scoring matrix: Blosum62, First Gap: 12, Extending Gap:2. The sequence identity (in %) between the DEAD-box RNA helicasepolypeptide sequences useful in performing the methods of the inventioncan be as low as 45% (is generally higher than 45%) compared to SEQ IDNO: 2.

TABLE B1 Description of proteins in FIG. 4 DEAD-box RNA helicaseProtein. Plant Source_Chomosome locus  1. A.thaliana_AT1G31970  2.Aquilegia_sp_TC27171  3. Chlorella_36420  4. G.max_Glyma01g01390  5.G.max_GM06MC17187_59654952@16891#1_A  6. G.raimondii_TC4727  7.H.vulgare_TC183460  8. M.truncatula_CU459038_20  9. O.lucimarinus_3808410. O.RCC809_33138 11. O.sativa_LOC_Os07g20580 12. O.taurii_19637 13.P.patens_205380 14. S.bicolor_Sb02g009590 15. V.carter_103394 16.V.vinifera_GSVIVT00003606001 17. Z.mays_ZM07MC20090_BFb0116P06@20039#1_A

2. CER2-Like Polypeptides

Results of the software analysis are shown in FIG. 10 for the globalsimilarity and identity over the full length of the polypeptidesequences. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line. Parameters used in the comparison were: Scoringmatrix: Blosum62, First Gap: 12, Extending Gap: 2. The sequence identity(in %) between the CER2-like polypeptide sequences useful in performingthe methods of the invention is generally higher than 16% compared toSEQ ID NO: 169.

3. At1g68440-Like Polypeptides

Results of the software analysis are shown in FIG. 15 for the globalsimilarity and identity over the full length of the polypeptidesequences. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line. Parameters used in the comparison were: Scoringmatrix: Blosum62, First Gap: 12, Extending Gap: 2. The sequence identity(in %) between the At1g68440-like polypeptide sequences useful inperforming the methods of the invention can be as low as 21% compared toSEQ ID NO: 292.

Example 4 Identification of Domains Comprised in Polypeptide SequencesUseful in Performing the Methods of the Invention

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

1. DEAD-Box RNA Helicase Polypeptide

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 2 are presented in Table C1.

TABLE C1 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 2. Amino acidcoordinates on SEQ ID NO 2: e-value [amino Interpro ID Domain ID Domainname acid position of the domain] IPR000629 PS00039 RNA helicase, ATP-0.0 [246-254]T PROSITE dependent, DEAD-box, conserved site IPR001650PF00271 DNA/RNA helicase, C- 7.8E−31 [361-437]T PFAM0 terminal SM00490DNA/RNA helicase, C- 1.9E−30 [356-437]T SMART terminal PS51194 DNA/RNAhelicase, C- 0.0 [333-476]T PROFILE terminal IPR0011545 PF00270 DNA/RNAhelicase, 8.3E−65 [116-289]T PFAM DEAD/DEAH box type, N-terminalIPR014001 SM00487 DEAD-like helicase, N- 8.6E−66 [111-317]T SMARTterminal IPR014014 PS51195 RNA helicase, DEAD- 0.0 [94-120]T PROFILE boxtype, Q motif IPR014021 PS51192 Helicase, superfamily 1 0.0 [123-300]TPROFILE and 2, ATP-binding unintegrated G3DSA:3.40.50.300 unintegrated2.9E−64 [81-311]T GENE3D 4.2E−42 [311-477]T PTHR10967 unintegrated 0.0[92-494]T PANTHER 0.0 [92-494]T PTHR10967:SF50 unintegrated 0.0[92-494]T PANTHER 0.0 [92-494]T SSF52540 unintegrated 2.7E−60 [11-457]TSUPERFAMILY 6.2E−43 [310-472]T

2. CER2-Like Polypeptides

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 169 are presented in Table C2.

TABLE C2 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 169. Interpro IDDomain ID Domain name Short Name Location IPRO003480 PF02458 TransferaseTransferase 8.6E−15 [73-189]T PFAM unintegrated G3DSA:3.30.559.20unintegrated G3DSA:3.30.559.20 9.6E−31 [16-192]T GENE3D3. At1g68440-Like Polypeptides

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 292 are presented in Table C3.

TABLE C2 InterPro scan results of the polypeptide sequence asrepresented by SEQ ID NO: 292. Protein Transmembrane Amino acidcoordinates length region E-value on SEQ ID NO 292 332 TMHMM NA Start:46; Stop: 64

Example 5 Topology Prediction of the Polypeptide Sequences Useful inPerforming the Methods of the Invention

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters can be selected, such as organism group(non-plant or plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark    -   PSORT (URL: psort.org)    -   PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

Example 6 Assay Related to the Polypeptide Sequences Useful inPerforming the Methods of the Invention 1. DEAD-Box RNA HelicasePolypeptide

Tools and techniques for measuring helicase activity are e.g. describedin Okanami et al. (1998) Nucleic Acid Res. (26): 2638-2643.

2. CER2-Like Polypeptides

A radioisotopic assay for DAT can be adapted from De Luca et al., 1985,J. Plant Physiol. Vol 121, pages 417-428. The assay buffer which cancontain DTT can be replaced with Buffer B containing ascorbate,resulting in DAT enzyme assays with decreased background radioactivity.The assay mixture can contain 10 μl of protein solution (of variousconcentrations), 18 μM deacetylvindoline, and 37 μM[1-14Cjacetylcoenzyme A (sp act 54 mCi/mmol) and Buffer B in a finalvolume of 100 μl. Assays can be performed in 1.5 ml Eppendorf micro testtubes. After 10 min in a 30° C. water bath, the reaction can be stoppedwith 100 μl of 0.1 M NaOH. The radiolabeled product ([14C]vindoline) canbe extracted with 250 μl of ethyl acetate for 5 min on an EppendorfModel 5432 mixer. The aqueous and organic phases can be separated bycentrifugation for 2 min in an Eppendorf Model 235C microcentrifuge at13,000 rpm. One hundred microliters of the organic phase from each assaycan be mixed with 2.5 ml of 0.4% PPO in toluene and assays can becounted for 1 min using an LKB 1219 Rackbeta liquid scintillationcounter.

Example 7 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention 1. DEAD-Box RNA Helicase Polypeptide

The Oryza sativa DEAD-box RNA helicase nucleic acid sequence wasamplified by PCR using as template an Oryza sativa seedlings cDNAlibrary (Invitrogen, Paisley, UK). After reverse transcription of RNAextracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0.Average insert size of the bank was 1.5 kb and the original number ofclones was of the order of 1.59×107 cfu. Original titer was determinedto be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. Afterplasmid extraction, 200 ng of template was used in a 50 μl PCR mix.Primers prm14820 (SEQ ID NO: 157; sense: 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgggtcgcagtatgcttc-3′) and prm14821 (SEQ ID NO: 158;reverse, complementary:5′-ggggaccactttgtacaagaaagctgggtacgaagaaaagaccagcaaat-3′), which includethe AttB sites for Gateway recombination, were used for PCRamplification. PCR was performed using Hifi Taq DNA polymerase instandard conditions. A PCR fragment of the expected size was amplifiedand purified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombines in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”. Plasmid pDONR201was purchased from Invitrogen, as part of the Gateway® technology.

The entry clone comprising SEQ ID NO: 1 was then used in an LR reactionwith a destination vector used for Oryza sativa transformation. Thisvector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination (such that thesequence of interest from the entry clone is integrated in sense or antisense orientation) with the nucleic acid sequence of interest alreadycloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 156) forconstitutive specific expression was located upstream of this Gatewaycassette.

After the LR recombination step, the resulting expression vectorpGOS2::DEAD-box RNA helicase (FIG. 5) was transformed into Agrobacteriumstrain LBA4044 according to methods well known in the art.

2. CER2-Like Polypeptides

The nucleic acid sequence was amplified by PCR using as template acustom-made Medicago truncatula seedlings cDNA library. PCR wasperformed using Hifi Taq DNA polymerase in standard conditions, using200 ng of template in a 50 μl PCR mix. The primers used were prm13662(SEQ ID NO: 272; sense): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgggttcattgccaaattta-3′ and prm13663 (SEQ ID NO: 273;reverse, complementary):5′-ggggaccactttgtacaagaaagctgggtcacaacacaattccaccaact-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombined in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pCER2-like.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 168 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 289) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::CER2-like (FIG. 11) was transformed into Agrobacterium strainLBA4044 according to methods well known in the art.

3. At1g68440-Like Polypeptides

The nucleic acid sequence was amplified by PCR using as template acustom-made Populus trichocarpa seedlings cDNA library. PCR wasperformed using Hifi Taq DNA polymerase in standard conditions, using200 ng of template in a 50 μl PCR mix. The primers used were prm14292:(SEQ ID NO: 361; sense): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggagatcccagtgatcaat-3′ and prm14293: (SEQ ID NO: 362;reverse, complementary):5′-ggggaccactttgtacaagaaagctgggtccgcgttatcaaacagattt-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombined in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pAt1g68449.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 291 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 363) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::At1g68440-like (FIG. 16) was transformed into Agrobacteriumstrain LBA4044 according to methods well known in the art.

Example 8 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Example 9 Transformation of Other Crops Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M patent U.S. Pat. No. 5,164,310. Severalcommercial soybean varieties are amenable to transformation by thismethod. The cultivar Jack (available from the Illinois Seed foundation)is commonly used for transformation. Soybean seeds are sterilised for invitro sowing. The hypocotyl, the radicle and one cotyledon are excisedfrom seven-day old young seedlings. The epicotyl and the remainingcotyledon are further grown to develop axillary nodes. These axillarynodes are excised and incubated with Agrobacterium tumefacienscontaining the expression vector. After the cocultivation treatment, theexplants are washed and transferred to selection media. Regeneratedshoots are excised and placed on a shoot elongation medium. Shoots nolonger than 1 cm are placed on rooting medium until roots develop. Therooted shoots are transplanted to soil in the greenhouse. T1 seeds areproduced from plants that exhibit tolerance to the selection agent andthat contain a single copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7 Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Cotton Transformation

Cotton is transformed using Agrobacterium tumefaciens according to themethod described in U.S. Pat. No. 5,159,135. Cotton seeds are surfacesterilised in 3% sodium hypochlorite solution during 20 minutes andwashed in distilled water with 500 μg/ml cefotaxime. The seeds are thentransferred to SH-medium with 50 μg/ml benomyl for germination.Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cmpieces and are placed on 0.8% agar. An Agrobacterium suspension (approx.108 cells per ml, diluted from an overnight culture transformed with thegene of interest and suitable selection markers) is used for inoculationof the hypocotyl explants. After 3 days at room temperature andlighting, the tissues are transferred to a solid medium (1.6 g/lGelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg etal., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/mlcefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria.Individual cell lines are isolated after two to three months (withsubcultures every four to six weeks) and are further cultivated onselective medium for tissue amplification (30° C., 16 hr photoperiod).Transformed tissues are subsequently further cultivated on non-selectivemedium during 2 to 3 months to give rise to somatic embryos. Healthylooking embryos of at least 4 mm length are transferred to tubes with SHmedium in fine vermiculite, supplemented with 0.1 mg/l indole aceticacid, 6 furfurylaminopurine and gibberellic acid. The embryos arecultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the2 to 3 leaf stage are transferred to pots with vermiculite andnutrients. The plants are hardened and subsequently moved to thegreenhouse for further cultivation.

Example 10 Phenotypic Evaluation Procedure 10.1 Evaluation Setup 1.DEAD-Box RNA Helicase Polypeptide

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

2. CER2-Like Polypeptides

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Five events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%. Plantsgrown under non-stress conditions are watered at regular intervals toensure that water and nutrients are not limiting and to satisfy plantneeds to complete growth and development.

From the stage of sowing until the stage of maturity the plants arepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

T1 events can be further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation, e.g. with lessevents and/or with more individuals per event.

3. At1g68440-Like Polypeptides

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%. Plantsgrown under non-stress conditions were watered at regular intervals toensure that water and nutrients were not limiting and to satisfy plantneeds to complete growth and development.

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

T1 events can be further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation, e.g. with lessevents and/or with more individuals per event.

Drought Screen 1. DEAD-Box RNA Helicase Polypeptide

Plants from T1 seeds were grown in potting soil under normal conditionsuntil they approached the heading stage. They were then transferred to a“dry” section where irrigation was withheld. Humidity probes wereinserted in randomly chosen pots to monitor the soil water content(SWC). When SWC went below certain thresholds, the plants wereautomatically re-watered continuously until a normal level was reachedagain. The plants were then re-transferred again to normal conditions.The rest of the cultivation (plant maturation, seed harvest) was thesame as for plants not grown under abiotic stress conditions. Growth andyield parameters were recorded as detailed for growth under normalconditions.

2. CER2-Like Polypeptides

Plants from T2 seeds were grown in potting soil under normal conditionsuntil they approached the heading stage. They were then transferred to a“dry” section where irrigation was withheld. Humidity probes wereinserted in randomly chosen pots to monitor the soil water content(SWC). When SWC went below certain thresholds, the plants wereautomatically re-watered continuously until a normal level was reachedagain. The plants were then re-transferred again to normal conditions.The rest of the cultivation (plant maturation, seed harvest) was thesame as for plants not grown under abiotic stress conditions. Growth andyield parameters were recorded as detailed for growth under normalconditions.

3. At1g68440-Like Polypeptides

Plants from T2 seeds are grown in potting soil under normal conditionsuntil they approached the heading stage. They are then transferred to a“dry” section where irrigation is withheld. Humidity probes are insertedin randomly chosen pots to monitor the soil water content (SWC). WhenSWC goes below certain thresholds, the plants are automaticallyre-watered continuously until a normal level is reached again. Theplants are then re-transferred again to normal conditions. The rest ofthe cultivation (plant maturation, seed harvest) is the same as forplants not grown under abiotic stress conditions. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Nitrogen Use Efficiency Screen

Rice plants from T1 or T2 seeds are grown in potting soil under normalconditions except for the nutrient solution. The pots are watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) isthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Salt Stress Screen

Plants are grown on a substrate made of coco fibers and argex (3 to 1ratio). A normal nutrient solution is used during the first two weeksafter transplanting the plantlets in the greenhouse. After the first twoweeks, 25 mM of salt (NaCl) is added to the nutrient solution, until theplants are harvested. Seed-related parameters are then measured.

10.2 Statistical Analysis: F Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF test. A significant F test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

10.3 Parameters Measured

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles as described inWO2010/031780. These measurements were used to determine differentparameters.

Biomass-Related Parameter Measurement

The plant aboveground area or leafy biomass was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the area measured at the time point at which theplant had reached its maximal leafy biomass.

The early vigour is the plant (seedling) aboveground area three weekspost-germination. Increase in root biomass is expressed as an increasein total root biomass (measured as maximum biomass of roots observedduring the lifespan of a plant); or as an increase in the root/shootindex (measured as the ratio between root mass and shoot mass in theperiod of active growth of root and shoot).

Increase in root biomass is expressed as an increase in total rootbiomass (measured as maximum biomass of roots observed during thelifespan of a plant); or as an increase in the root/shoot index, whichis measured as the ratio between root mass and shoot mass in the periodof active growth of root and shoot. Root biomass can be determined usinga method as described in WO 2006/029987.

Parameters Related to Development Time

The early vigour is the plant, i.e. seedling, aboveground area threeweeks post-germination. Early vigour was determined by counting thetotal number of pixels from aboveground plant parts discriminated fromthe background. This value was averaged for the pictures taken on thesame time point from different angles and was converted to a physicalsurface value expressed in square mm by calibration.

AreaEmer is an indication of quick early development (when decreasedcompared to control plants). It is the ratio (expressed in %) betweenthe time a plant needs to make 30% of the final biomass and the time aplant needs to make 90% of its final biomass.

The “flowering time” of the plant can be determined using the method asdescribed in WO 2007/093444.

Seed-Related Parameter Measurements

The mature primary panicles were harvested, counted, bagged,barcode-labelled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed weight, was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand KernelWeight, or TKW, is extrapolated from the number of filled seeds countedand their total weight. The Harvest Index, or HI, in the presentinvention is defined as the ratio between the total seed yield and theabove ground area (mm²), multiplied by a factor 10⁶. The total number offlowers per panicle as defined in the present invention is the ratiobetween the total number of seeds and the number of mature primarypanicles. The seed fill rate as defined in the present invention is theproportion (expressed as a %) of the number of filled seeds over thetotal number of seeds (or florets).

Root biomass can be determined using a method as described in WO2006/029987.

Example 11 Results of the Phenotypic Evaluation of the TransgenicPlants 1. DEAD-Box RNA Helicase Polypeptide

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 1 under drought conditions are presentedbelow. See previous Examples for details on the generations of thetransgenic plants.

The results of the evaluation of transgenic rice plants under droughtstress conditions are presented below. An increase of at least 5% wasobserved for total seed yield including total weight of seeds, number offilled seeds, fill rate, and harvest index, and of more than 2.5% forthousand kernel weight.

The results of the evaluation of transgenic rice plants expressing aDEAD-box RNA helicase nucleic acid under drought-stress conditions arepresented hereunder (Table D1).

Example

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid encoding the DEAD-box RNAhelicase polypeptide of SEQ ID NO: 2 under drought stress conditions arepresented below in Table D1. When grown under drought stress conditions,An increase of at least 5% was observed for total seed yield includingtotal weight of seeds, number of filled seeds, fill rate, and harvestindex.

TABLE D1 Data summary for transgenic rice plants on drought; for eachparameter, the overall percent increase is shown if the parameterreaches a p-value of p < 0.05 and abovethe 5% treshold. ParameterOverall increase Total weight of seeds 23.2 Number of filled seeds 19.1Fill rate 35.0 Harvest index 25.3

2. CER2-Like Polypeptides

The results of the evaluation of transgenic rice plants expressing anCER2-like nucleic acid under drought-stress conditions are presentedhereunder. An increase of at least 5% was observed for total seedweight, number of filled seeds, fill rate, harvest index, and gravitycentre of the leafy biomass of a plant (Table D2). AreaCycl is a measureof the time elapse between sowing and the emergence of the first panicleof a plant, that is, the period of accruing biomass of a plant. It iscalculated as the time (in days) needed between sowing and the day theplant reaches 90% of its final biomass. It is typically used as ameasure for estimating the growth rate of a plant.

TABLE D2 Data summary for transgenic rice plants; for each parameter,the overall percent increase is shown for the confirmation (T1generation), for each parameter the p-value is <0.05. Parameter Overallincrease totalwgseeds 52.1 fillrate 54.2 harvestindex 49.7 nrfilledseed46.9 GravityYMax 5.3 AreaCycl 5.43. At1g68440-Like Polypeptides

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid comprising the longest OpenReading Frame in SEQ ID NO: 291 under non-stress conditions arepresented below. See previous Examples for details on the generations ofthe transgenic plants.

The results of the evaluation of transgenic rice plants under non-stressconditions are presented below. An increase of at least 5% was observedfor total seed yield, number of filled seeds, fill rate, number offlowers per panicle, harvest index, total seed weight, and of (2.5-3)%for thousand kernel weight

The results of the evaluation of transgenic rice plants expressing anAt1g68440-like nucleic acid under drought-stress conditions arepresented hereunder. An increase was observed for total seed weight,number of filled seeds, fill rate, harvest index and thousand-kernelweight (Table D3).

The results of the evaluation of transgenic rice plants in the T1generation and expressing a nucleic acid encoding the At1g68440-likepolypeptide of SEQ ID NO: 292 under non-stress conditions are presentedbelow in Table D3. When grown under non-stress conditions, an increaseof at least 5% was observed for seed yield (total weight of seeds,number of filled seeds, fill rate, harvest index).

TABLE D3 Data summary for transgenic rice plants; for each parameter,the overall percent increase is shown for the confirmation (T1generation), for each parameter the p-value is <0.05. Parameter Overallincrease totalwgseeds 38.1 fillrate 28.5 harvestindex 34.4 nrfilledseed32.7

MEGA

1-85. (canceled)
 86. A method for enhancing yield-related traits in aplant relative to a control plant, comprising: (a) modulating expressionin a plant of a nucleic acid encoding a CER2-like polypeptide, whereinsaid CER2-like polypeptide comprises a PF02458 domain and/or possessestransferase activity; or (b) modulating expression in a plant of anucleic acid encoding an At1g68440-like polypeptide, wherein saidAt1g68440-like polypeptide comprises a transmembrane domain; or (c)reducing or substantially eliminating expression in a plant of a nucleicacid encoding a DEAD-box RNA helicase polypeptide, and/or level and/oractivity of a DEAD-box RNA helicase polypeptide in said plant, whereinsaid DEAD-box RNA helicase polypeptide comprises signature pattern[LIVMF]-[LIVMFFD-E-A-D4RKEN]-X-[LIVMFYGSTN] (SEQ ID NO: 159).
 87. Themethod of claim 86, wherein said modulated expression is effected byintroducing and expressing in a plant said nucleic acid encoding aCER2-like polypeptide; or wherein said modulated expression is effectedby introducing and expressing in a plant said nucleic acid encoding anAt1g68440-like polypeptide; or wherein said reduction or substantialelimination of expression and/or level and/or activity comprisesintroducing into a host plant a DEAD-box RNA helicase nucleic acid or afragment thereof substantially homologous to said nucleic acid encodinga DEAD-box RNA helicase polypeptide.
 88. The method of claim 86, whereinsaid enhanced yield-related traits comprise increased yield relative toa control plant, and preferably comprise increased biomass and/orincreased seed yield relative to a control plant, and/or wherein saidincreased seed yield obtained by reducing or substantially eliminatingexpression and/or level and/or activity in said plant is selected fromone or more of the following: a) increased seed biomass, i.e. seedweight; b) increased number of filled seeds; c) increased fill rate; andd) increased harvest index.
 89. The method of claim 86, wherein saidenhanced yield-related traits are obtained under non-stress conditions,or wherein said enhanced yield-related traits are obtained underconditions of drought stress, salt stress or nitrogen deficiency. 90.The method of claim 86, wherein said CER2-like polypeptide comprises oneor more of the following motifs: Motif 18a: (SEQ ID NO: 274)FKCGGL[SA][LI]G[LV][SG]W[AS]HI[LV][GA]D[GA]FSA SHFIN[SA]W, Motif 19a:(SEQ ID NO: 276) SGR[PL]E[IVL]KCNDEG[VA][RL][FI][VI]EAE[CA]D, Motif 20a:(SEQ ID NO: 278) xY[ST][TR][FY]E[AI]L[AST][AG][HL][IV]W[RK][CS]I[AC]KARG;

or wherein said CER2-like polypeptide comprises: Motif 21a:(SEQ ID NO: 280) VQ[VF]TxFKCGG[LM][SA][LIV]GLS[WC]AH[IVL]LGD[APV]FSA[ST]TF[FIM][NK]KW, Motif 22a: (SEQ ID NO: 282)[EA]SGR[PW][YF][IV]KCND[AC]GVRIVEA[KHR]CDK, Motif 23a: (SEQ ID NO: 284)SR[VT][GE][EP][GN]Kx[HY]E[LP][ST]x[LM]DLAMKLHY [LVI]R[GA]VY[FY][FY];

or wherein said CER2-like polypeptide comprises: Motif 24:(SEQ ID NO: 286) FKCGG[VIF][SA][LI]G[VL]G[MI]SHx[VLM]ADGxSALHFI[NS][SAT]W, Motif 25: (SEQ ID NO: 287)NPNLL[IV][TV]SWT[RT][LF]P[ILF][YH][DE]ADFGWG [KR]PI[FY]MGP, Motif 26:(SEQ ID NO: 288) [SA]LS[KE][VT]L[VT][HP][FY]YP[ML]AGR.


91. The method of claim 86, wherein said At1g68440-like polypeptidecomprises one or more of the following motifs: Motif 27a:(SEQ ID NO: 364) Y[SN]F[WC][KT]WGALILA[LV][FVL]A, Motif 28a:(SEQ ID NO: 366) ME[IV][PT][VE][IL]N[RL]I[SG]DF, Motif 29a:(SEQ ID NO: 368) [SN]VV[KQ]LWD[SN]LG[LF].


92. The method of claim 86, wherein said DEAD-box RNA helicasepolypeptide is an orthologue or paralogue to subfamily VI of the 6subfamilies of DEAD-box RNA helicases as described by Aubourg et al.(1999).
 93. The method of claim 86, wherein said DEAD-box RNA helicasepolypeptide comprises one or more of the following motifs: (i) Motif 1:(SEQ ID NO: 3) [LM][VI]ATDVA[AS]RGLD[IV][KP]D[VI][EK]VV[IV]N[YF][SD][YF]P[LN][TD][IT][ED]DYVHRIGRTGRA, (ii) Motif 2: (SEQ ID NO: 4)[RSN]L[NR][DR]V[TS][YF][LV]VLDEADRMLDMGFEP [EQ][IV]R[AK]I[VL], (iii)Motif 3: (SEQ ID NO: 5) P[TS]PIQA[YQ][AS][WI]P[YI][AL][LM][DS]GRD[FL][IV][GA]IA[KA]TGSGKT.


94. The method of claim 86, wherein said nucleic acid encoding aCER2-like polypeptide is of plant origin, from a dicotyledonous plant,from the family Fabaceae, from the genus Medicago, or from Medicagotruncatula; or wherein said nucleic acid encoding an At1g68440-like isof plant origin, from a dicotyledonous plant, from the familySalicaceae, from the genus Populus, or from Populus trichocarpa; orwherein said introduced DEAD-box RNA helicase-encoding nucleic acid isfrom the same family, from the same genus, or from the same species asthe host plant.
 95. The method of claim 86, wherein said nucleic acidencoding a CER2-like polypeptide encodes any one of the polypeptideslisted in Table A2 or is a portion of such a nucleic acid, or a nucleicacid capable of hybridizing with such a nucleic acid; or wherein saidnucleic acid encoding an At1g68440-like encodes any one of thepolypeptides listed in Table A3 or is a portion of such a nucleic acid,or a nucleic acid capable of hybridizing with such a nucleic acid; orwherein said nucleic acid encoding a DEAD-box RNA helicase polypeptideencodes any one of the proteins listed in Table A1 or is a portion ofsuch a nucleic acid, or a nucleic acid capable of hybridizing with sucha nucleic acid.
 96. The method of claim 86, wherein said nucleic acidencoding a CER2-like polypeptide encodes an orthologue or paralogue ofany of the polypeptides given in Table A2; or wherein said nucleic acidencoding an At1g68440-like polypeptide encodes an orthologue orparalogue of any of the polypeptides given in Table A3; or wherein saidnucleic acid encoding a DEAD-box RNA helicase polypeptide sequenceencodes an orthologue or paralogue of any of the proteins given in TableA1.
 97. The method of claim 86, wherein said nucleic acid encoding anAt1g68440-like polypeptide corresponds to SEQ ID NO: 291; or whereinsaid nucleic acid encoding a CER2-like polypeptide corresponds to SEQ IDNO: 168; or wherein said introduced DEAD-box RNA helicase nucleic acidsequence comprises a sufficient length of substantially contiguousnucleotides of SEQ ID NO: 1 or an orthologue or paralogue thereof andwherein said host plant is a cereal, preferably rice.
 98. The method ofclaim 97, wherein said nucleic acid encodes a DEAD-box RNA helicasepolypeptide is represented by SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155,and/or wherein said nucleic acid encoding a DEAD-box RNA helicasepolypeptide is represented by SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152 or 154.99. The method of claim 86, wherein said nucleic acid encoding aCER2-like polypeptide or said nucleic acid encoding an At1g68440-likepolypeptide is operably linked to a constitutive promoter, a mediumstrength constitutive promoter, a plant promoter, a GOS2 promoter, or aGOS2 promoter from rice; or wherein said reduction or substantialelimination of expression in a plant of a nucleic acid encoding aDEAD-box RNA helicase polypeptide or of the level and/or the activity ofa DEAD-box RNA helicase polypeptide is effected in a constitutive mannerby using a constitutive promoter, a GOS2 promoter, or a GOS2 promoterfrom rice.
 100. The method of claim 86, wherein said reduction orsubstantial elimination is effected by RNA-mediated downregulation ofgene expression, by co-suppression, or by use of antisense DEAD-box RNAhelicase nucleic acid sequences; or wherein said reduction orsubstantial elimination is effected by using an inverted repeat of aDEAD-box RNA helicase nucleic acid sequences or fragment thereof; orwherein said reduction or substantial elimination is effected using amicroRNA; or wherein said reduction or substantial elimination iseffected by insertion mutagenesis.
 101. A pant, plant part, includingseeds, or plant cell, obtained by the method of claim 86, wherein saidplant, plant part or plant cell, or seeds, comprises a recombinantnucleic acid encoding a CER2-like polypeptide, an At1g68440-likepolypeptide or a DEAD-box RNA helicase polypeptide as defined in claim86.
 102. A construct comprising: (i) the nucleic acid encoding aCER2-like polypeptide or the nucleic acid encoding an At1g68440-likepolypeptide as defined in claim 86, or an inverted repeat of a DEAD-boxRNA helicase gene or fragment thereof, capable of downregulating anendogenous target DEAD-box RNA helicase; (ii) one or more controlsequences capable of driving expression of the nucleic acid sequence of(i); and optionally (iii) a transcription termination sequence.
 103. Theconstruct of claim 102, wherein one of said control sequences is aconstitutive promoter, a medium strength constitutive promoter, a plantpromoter, a GOS2 promoter, or a GOS2 promoter from rice.
 104. A methodfor the production of a transgenic plant having enhanced yield-relatedtraits relative to a control plant, preferably increased yield relativeto a control plant, and more preferably increased seed yield and/orincreased biomass relative to a control plant, comprising: (i)introducing and expressing in a plant or plant cell the construct ofclaim 102; and (ii) cultivating said plant or plant cell underconditions promoting plant growth and development.
 105. A plant, plantpart or plant cell transformed with the construct of claim
 102. 106. Atransgenic plant or a plant cell derived from said transgenic plant,wherein said transgenic plant has enhanced yield-related traits relativeto a control plant, preferably increased yield relative to a controlplant, and more preferably increased seed yield and/or increasedbiomass, resulting from modulated expression of a nucleic acid encodinga CER2-like polypeptide as defined in claim 86; or wherein saidtransgenic plant has enhanced yield-related traits relative to a controlplant, preferably increased yield relative to a control plant, and morepreferably increased seed yield and/or increased biomass, resulting frommodulated expression of a nucleic acid encoding an At1g68440-likepolypeptide as defined in claim 86; or wherein said transgenic plant hasincreased yield, particularly increased biomass and/or increased seedyield, relative to a control plant, resulting from reduced orsubstantially eliminated expression of a nucleic acid encoding aDEAD-box RNA helicase polypeptide as defined in claim
 86. 107. The plantof claim 101, or a plant cell derived therefrom, wherein said plant is acrop plant, such as beet, sugarbeet or alfalfa; or a monocotyledonousplant such as sugarcane; or a cereal, such as rice, maize, wheat,barley, millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn,teff, milo or oats.
 108. Harvestable parts of the plant of claim 101,wherein said harvestable parts are preferably shoot biomass and/or seedsand/or products derived from said plants and/or from harvestable partsthereof.
 109. The method of claim 104, wherein the plant expresses thenucleic acid encoding a CER2-like polypeptide has enhanced yield-relatedtraits relative to a control plant, preferably increased yield, and morepreferably increased seed yield and/or increased biomass relative to acontrol plant; or wherein the plant expresses the nucleic acid encodingan At1g68440-like polypeptide has enhanced yield-related traits relativeto a control plant, preferably increased yield, and more preferablyincreased seed yield and/or increased biomass relative to a controlplant; or wherein the plant expresses the DEAD-box RNA helicase gene orfragment thereof for the reduction or substantial elimination of anendogenous target DEAD-box RNA helicase has increased yield relative toa control plant, preferably wherein said increased yield is increasedseed yield, further preferably wherein said increased seed yield isselected from one or more of the following: a) increased seed biomass,i.e. seed weight; b) increased number of filled seeds; c) increased fillrate and d) increased harvest index and/or wherein said yield increaseoccurs under stress conditions.
 110. An isolated nucleic acid moleculeselected from the group consisting of: (i) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 291; (ii) a nucleicacid molecule comprising the complement of the nucleotide sequence ofSEQ ID NO: 291; (iii) a nucleic acid molecule encoding an At1g68440-likepolypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO: 292, andadditionally or alternatively comprising one or more motifs having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to any one or more of the motifs given inSEQ ID NO: 364 to SEQ ID NO: 375, and further preferably conferringenhanced yield-related traits relative to control plants; (iv) a nucleicacid molecule which hybridizes with any of the nucleic acid molecules of(i) to (iii) under high stringency hybridization conditions andpreferably confers enhanced yield-related traits relative to controlplants; and (v) a nucleic acid molecule encoding an At1g68440-likepolypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO: 292 and anyof the other amino acid sequences in Table A3 and preferably conferringenhanced yield-related traits relative to control plants.
 111. Anisolated polypeptide selected from the group consisting of: (i) apolypeptide comprising the amino acid sequence of SEQ ID NO: 292; (ii) apolypeptide comprising an amino acid sequence having at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%; 95%, 96%, 97%, 98% or 99% sequence identity to the amino acidsequence of SEQ ID NO: 292, and additionally or alternatively comprisingone or more motifs having in increasing order of preference at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to any one or more of the motifs given in SEQ IDNO: 364 to SEQ ID NO: 375, and/or any of the other amino acid sequencesin Table A3, and further preferably conferring enhanced yield-relatedtraits relative to control plants; and (iii) derivatives of thepolypeptide of (i) or (ii) above.
 112. An isolated nucleic acid moleculeselected from the group consisting of: (a) a nucleic acid moleculecomprising the polynucleotide sequence of SEQ ID NO: 12 or 14; (b) anucleic acid molecule comprising the complement of the polynucleotidesequence of SEQ ID NO: 12 or 14; (c) a nucleic acid molecule encoding aDEAD-box RNA helicase polypeptide having at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQID NO: 13 or 15, and additionally or alternatively comprising one ormore motifs having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more ofthe motifs given in SEQ ID NO: 3 to SEQ ID NO: 11, and furtherpreferably conferring enhanced yield-related traits relative to controlplants; and (d) a nucleic acid molecule which hybridizes with any of thenucleic acid molecules of (a) to (c) under high stringency hybridizationconditions and preferably confers enhanced yield-related traits relativeto control plants.
 113. An isolated polypeptide selected from the groupconsisting of: (i) a polypeptide comprising the amino acid sequence ofSEQ ID NO: 13 or 15; (ii) a polypeptide comprising an amino acidsequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO: 13 or 15, andadditionally or alternatively comprising one or more motifs having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to any one or more of the motifs given inSEQ ID NO: 3 to SEQ ID NO: 11, and further preferably conferringenhanced yield-related traits relative to control plants; and (iii)derivatives of the polypeptide of (i) or (ii) above.